Bacillus thuringiensis CryET33 and CryET34 compositions and uses therefor

ABSTRACT

Disclosed are Bacillus thuringiensis strains comprising novel crystal proteins which exhibit insecticidal activity against coleopteran insects including red flour beetle larvae (Tribolium castaneum) and Japanese beetle larvae (Popillia japonica). Also disclosed are novel B. thuringiensis crystal toxin genes, designated cryET33 and cryET34, which encode respectively the coleopteran-toxic proteins, CryET33 (29-kDa) crystal protein, and CryET34 (14-kDa) crystal protein. Also disclosed are methods of making and using transgenic cells comprising the novel nucleic acid sequences of the invention.

The present application is a continuation-in-part application based onU.S. Pat. Ser. No. 08/718,905, filed Sep. 24, 1996, (U.S. Pat. No.6,063,756) the entire contents of which is specifically incorporatedherein by reference in its entirety.

1. BACKGROUND OF THE INVENTION

1.1 Field of the Invention

The present invention relates generally to the fields of molecularbiology. More particularly, certain embodiments concern methods andcompositions comprising DNA segments, and proteins derived frombacterial species. More particularly, it concerns novel cryET33 andcryET34 genes from Bacillus thuringiensis encoding coleopteran-toxiccrystal proteins. Various methods for making and using these DNAsegments, DNA segments encoding synthetically-modified Cry proteins, andnative and synthetic crystal proteins are disclosed, such as, forexample, the use of DNA segments as diagnostic probes and templates forprotein production, and the use of proteins, fusion protein carriers andpeptides in various immunological and diagnostic applications. Alsodisclosed are methods of making and using nucleic acid segments in thedevelopment of transgenic plant cells containing the DNA segmentsdisclosed herein.

1.2 Description of the Related Art

1.2.1 Bacillus Thuringiensis Crystal Proteins

One of the unique features of B. thuringiensis is its production ofcrystal proteins during sporulation which are specifically toxic tocertain orders and species of insects. Many different strains of B.thuringiensis have been shown to produce insecticidal crystal proteins.Compositions including B. thuringiensis strains which produce proteinshaving insecticidal activity against lepidopteran and dipteran insectshave been commercially available and used as environmentally-acceptableinsecticides because they are quite toxic to the specific target insect,but are harm less to plants and other non-targeted organisms.

The mechanism of insecticidal activity of the B. thuringiensis crystalproteins has been studied extensively in the past decade. It has beenshown that the crystal proteins are toxic to the insect only afteringestion of the protein by the insect. The alkaline pH and proteolyticenzymes in the insect mid-gut solubilize the proteins, thereby allowingthe release of components which are toxic to the insect. These toxiccomponents disrupt the mid-gut cells, cause the insect to cease feeding,and, eventually, bring about insect death. For this reason, B.thuringiensis has proven to be an effective and environmentally safeinsecticide in dealing with various insect pests.

As noted by Höfte et al., (1989) the majority of insecticidal B.thuringiensis strains are active against insects of the orderLepidoptera, i.e., caterpillar insects. Other B. thuringiensis strainsare insecticidally active against insects of the order Diptera, i.e.flies and mosquitoes, or against both lepidopteran and dipteran insects.In recent years, a few B. thuringiensis strains have been reported asproducing crystal proteins that are toxic to insects of the orderColeoptera, i.e., beetles (Krieg et al., 1983; Sick et al., 1990;Lambert et al., 1992).

1.2.2 Genetics of Crystal Proteins

A number of genes encoding crystal proteins have been cloned fromseveral strains of B. thuringiensis. The review by Höfte et al. (1989)discusses the genes and proteins that were identified in B.thuringiensis prior to 1990, and sets forth the nomenclature andclassification scheme which has traditionally been applied to B.thuringiensis genes and proteins. cryI genes encode lepidopteran-toxicCryI proteins. cryII genes encode CryII proteins that are toxic to bothlepidopterans and dipterans. cryIII genes encode coleopteran-toxicCryIII proteins, while cryIV genes encode dipteran-toxic CryIV proteins.

Recently a new nomenclature has been proposed which systematicallyclassifies the cry genes based upon DNA sequence homology rather thanupon insect specificities. This classification scheme is shown in Table1.

TABLE 1 REVISED B. THURINGIENSIS δ-ENDOTOXIN NOMENCLATUREA New OldGenBank Accession # Cry1Aa CryIA(a) M11250 Cry1Ab CryIA(b) M13898 Cry1AcCryIA(c) M11068 Cry1Ad CryIA(d) M73250 Cry1Ae CryIA(e) M65252 Cry1BaCryIB X06711 Cry1Bb ET5 L32020 Cry1Bc PEG5 Z46442 Cry1Bd CryE1 U70726Cry1Ca CryIC X07518 Cry1Cb CryIC(b) M97880 Cry1Da CryID X54160 Cry1DbPrtB Z22511 Cry1Ea CryIE X53985 Cry1Eb CryIE(b) M73253 Cry1Fa CryIFM63897 Cry1Fb PrtD Z22512 Cry1Ga PrtA Z22510 Cry1Gb CryH2 U70725 Cry1HaPrtC Z22513 Cry1Hb U35780 Cry1Ia CryV X62821 Cry1Ib CryV U07642 Cry1JaET4 L32019 Cry1Jb ET1 U31527 Cry1K U28801 Cry2Aa CryIIA M31738 Cry2AbCryIIB M23724 Cry2Ac CryIIC X57252 Cry3A CryIIIA M22472 Cry3Ba CryIIIBX17123 Cry3Bb CryIIIB2 M89794 Cry3C CryIIID X59797 Cry4A CryIVA Y00423Cry4B CryIVB X07423 Cry5Aa CryVA(a) L07025 Cry5Ab CryVA(b) L07026 Cry5BU19725 Cry6A CryVIA L07022 Cry6B CryVIB L07024 Cry7Aa CryIIIC M64478Cry7Ab CryIIICb U04367 Cry8A CryIIIE U04364 Cry8B CryIIIG U04365 Cry8CCryIIIF U04366 Cry9A CryIG X58120 Cry9B CryIX X75019 Cry9C CryIH Z37527Cry1OA CryIVC M12662 Cry11A CryIVD M31737 Cry11B Jeg80 X86902 Cry12ACryVB L07027 Cry13A CryVC L07023 Cry14A CryVD U13955 Cry15A 34kDa M76442Cry16A cbm7l X94146 Cry17A cbm7l X99478 Cry18A CryBP1 X99049 Cry19AJeg65 Y08920 Cyt1Aa CytA X03182 Cyt1Ab CytM X98793 Cyt1B U37196 Cyt2ACytB Z14147 Cyt2B CytB U52043 ^(a)Adapted from:http://epunix.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html

1.2.3 Identification of Crystal Proteins Toxic to Coleopteran Insects

The utility of bacterial crystal proteins as insecticides was extendedwhen the first isolation of a coleopteran-toxic B. thuringiensis strainwas reported (Kreig et al., 1983; 1984). This strain (described in U.S.Pat. No. 4,766,203, specifically incorporated herein by reference),designated B. thuringiensis var. tenebrionis, is reported to be toxic tolarvae of the coleopteran insects Agelastica alni (blue alder leafbeetle) and Leptinolarsa decemlineata (Colorado potato beetle).

U.S. Pat. No. 4,766,203 (specifically incorporated herein by reference)relates to a 65-70 kilodalton (kDa) insecticidal crystal proteinidentified in B. thuringiensis tenebrionis (see also Berhnard, 1986).Sekar et al., (1987) report the cloning and characterization of a genefor a coleopteran-toxic crystal protein from B. thuringiensistenebrionis. The predicted size of the polypeptide (as deduced from thegene sequence) is 73 kDa, however, the isolated protein consistsprimarily of a 65-kDa component. Höfte et al. (1987) also reports theDNA sequence for the cloned gene from B. thuringiensis tenebroinis, withthe sequence of the gene being identical to that reported by Sekar etal. (1987).

McPherson et al. (1988) discloses a DNA sequence for the cloned insectcontrol gene from B. thuringiensis tenebrionis, the sequence wasidentical to that reported by Sekar et al. (1987). E. coli cells andPseudomonas fluorescens cells harboring the cloned gene were found to betoxic to Colorado potato beetle larvae.

Intl. Pat. Appl. Publ. No. WO 91/07481 dated May 30, 1991, describes B.thuringiensis mutants that produce high yields of the same insecticidalproteins originally made by the parent strains at lesser yields. Mutantsof the coleopteran-toxic B. thuringiensis tenebrionis strain aredisclosed.

A coleopteran-toxic strain, designated B. thuringiensis var. san diego,was reported by Herrnstadt et al. (1986) to produce a 64-kDa crystalprotein toxic to some coleopterans, including Pyrrhalta luteola (elmleaf beetle); Anthonomus gradis (boll weevil), Leptinotarsa decemlineata(Colorado potato beetle), Osiorhynchus sulcatus (black vine weevil),Tenebrio molitor (yellow mealworm), Haltica zombacina; and Diabroticaundecimpunctata undecimpunctata (western spotted cucumber beetle).

The DNA sequence of a coleopteran toxin gene from B. thuringiensis sandiego was reported by Herrnstadt et al. (1987); and was disclosed inU.S. Pat. No. 4,771,131. The sequence of the toxin gene of B.thuringiensis san diego is identical to that reported by Sekar et al.(1987) for the cloned coleopteran toxin gene of B. thuringiensistenebrionis. Krieg et al., (1987) demonstrated that B. thuringiensis sandiego was identical to B. thuringiensis tenebrionis, based on variousdiagnostic tests.

Another B. thuringiensis strain, EG2158, was reported by Donovan et al.(1988) and described in U.S. Pat. No. 5,024, 837. EG2158 produces a73-kDa CryC crystal protein that is insecticidal to coleopteran insects.Its DNA sequence was identical to that reported by Sekar et al. (1987)for the cloned B. thuringiensis tenebrionis toxin gene. This coleopterantoxin gene is referred to as the cryIIIA gene by Höfte et al., 1989. Twominor proteins of 30- and 29-kDa were also observed in this strain, butwere not further characterized (Donovan et al., 1988).

U.S. Pat. No. 5,024, 837 also describes hybrid B. thuringiensis var.kurstaki stains which showed activity against both lepidopteran andcoleopteran insects. U.S. Pat. No. 4,797,279 (corresponding to EP0221024) discloses a hybrid B. thuringiensis transformed with a plasmidfrom B. thuringiensis var. kurstaki containing a lepidopteran-toxiccrystal protein-encoding gene and a plasmid from B. thuringiensistenebrionis containing a coleopteran-toxic crystal protein-encodinggene. The hybrid B. thuringiensis strain produces crystal proteinscharacteristic of those made by both B. thuringiensis kurstaki and B.thuringiensis tenebrionis. U.S. Pat. No. 4,910,016 (corresponding to EP0303379) discloses a B. thuringiensis isolate identified as B.thuringiensis MT 104 which has insecticidal activity againstcoleopterans and lepidopterans.

European Pat. Appl. Publ. No. 0318143 discloses an intact,partially-modified gene from B. thuringiensis tenebrionis andrecombinant vectors comprising it able to direct expression of a proteinhaving toxicity to coleopteran insects, and Eur. Pat. Appl. Publ. No.0324254 discloses B. thuringiensis A30; a strain which has insecticidalactivity against coleopteran insects, including Colorado potato beetlelarvae, corn rootworm larvae and boll weevils.

U.S. Pat. No. 4,999,192 (corresponding to EP 0328383) discloses B.thuringiensis PS40D1 which has insecticidal activity against Coloradopotato beetle larvae. The strain was also identified via serotyping asbeing serovar 8a8b, morrisoni. U.S. Pat. No. 5,006,336 (corresponding toEP 0346114) described a B. thuringiensis isolate, designated PS122D3,which was serotyped as serovar 8a8b, morrisoni and which exhibitedinsecticidal activity against Colorado potato beetle larvae. U.S. Pat.No. 4,966,765 (corresponding to EP 0330342) discloses a B. thuringiensisstrain, PS86B1 (identified via serotyping as being serovar tolworthi),which has insecticidal activity against the Colorado potato beetle.

The nucleotide sequence of a cryIIIB gene and its encodedcoleopteran-toxic protein is reported by Sick et al., (1990) but the B.thuringiensis source strain is identified only via serotyping as beingsubspecies tolworthi. U.S. Pat. No. 4,966,155, issued Feb. 26, 1991, ofSick et al. (corresponding to EP 0337604), discloses a B. thuringiensistoxin gene obtained from the coleopteran-active B. thuringiensis 43F,and the gene sequence appears identical to the cryIIIB gene. B.thuringiensis 43F is reported as being active against Colorado potatobeetle and Leptinotarsa texana.

Eur. Pat. Appl. Publ. No. 0382990 discloses two B. thuringiensisstrains, btPGS1208 and btPGS1245, which produce crystal proteins of 74-and 129-kDa, respectively, that exhibit insecticidal activity againstColorado potato beetle larvae. The DNA sequence reported for toxin geneproducing the 74-kDa protein appears to be related to that of thecryIIIB gene of Sick et al (1990).

PCT Intl. Pat. Appl. Publ. No. WO 90/13651 discloses B. thuringiensisstrains which contain a toxin gene encoding an 81-kDa protein that issaid to be toxic to both lepidopteran and coleopteran insects. U.S. Pat.No. 5,055,293 discloses the use of B. laterosporous for corn rootworm(Diabrotica) insect control.

2. SUMMARY OF THE INVENTION

In sharp contrast to the prior art, the novel coleopteran-active CryET33and CryET34 crystal proteins of the present invention and the novel DNAsequences which encode them represent a new class of B. thuringiensiscrystal proteins, and do not share sequence homology with any of thestrains described in the aforementioned literature. The B. thuringiensisisolate disclosed and claimed herein represents the first B.thuringiensis kurstaki strain that has been shown to be toxic tocoleopterans. The B. thuringiensis strains of the present inventioncomprise novel cry genes that express protein toxins having insecticidalactivity against coleopterans such as insects of the genera Popillia andTribolium.

One aspect of the present invention relates to novel nucleic acidsegments that comprise two coleopteran-toxin δ-endotoxin genes havingnucleotide base sequences and deduced amino acid sequences asillustrated in FIG. 1A, FIG. 1B, and FIG. 1C. Hereinafter, these genesare designated cryET33 (SEQ ID NO:1) and cryET34 (SEQ ID NO:2). ThecryET33 gene has a coding region extending from nucleotide bases 136 to939 shown in FIG. 1A, FIG. 1B, and FIG. 1C and the cryET34 gene has acoding region extending from nucleotide bases 969 to 1349 shown in FIG.1A, FIG. 1B, and FIG. 1C.

Another aspect of the present invention relates to the insecticidalproteins encoded by the novel cryET33 and cryET34 genes. The deducedamino acid sequence of the CryET33 protein (SEQ ID NO:3), encoded by thecryET33 gene from the nucleotide bases 136 to 936, is shown in FIG. 1A,FIG. 1B, and FIG. 1C. The deduced amino acid sequence of the CryET34protein (SEQ ID NO:4), encoded by the cryET34 gene from nucleotide bases969 to 1346, is also shown in FIG. 1A, FIG. 1B, and FIG. 1C. Theproteins exhibit insecticidal activity against insects of the orderColeoptera, in particular, boll weevil, red flour beetle and Japanesebeetle.

Another aspect of the present invention relates to a biologically-pureculture of a naturally occurring, wild-type B. thuringiensis bacterium,strain EG10327, deposited on Dec. 14, 1994 with the AgriculturalResearch Culture Collection, Northern Regional Research Laboratory(NRRL) having Accession No. NRRL B-21365. B. thuringiensis EG10327 isdescribed infra in sections 5.1-5.3. B. thuringiensis EG10327 is anaturally-occurring B. thuringiensis strain that contains genes whichare related to or identical with the cryET33 and cryET34 genes of thepresent invention. EG10327 produces 29-kDa and 14-kDa insecticidalproteins that are related to or identical with the CryET33 and CryET34proteins disclosed herein.

Another aspect of the present invention relates to a recombinant vectorcomprising one or both of the novel cryET33 and cryET34 genes, arecombinant host cell transformed with such a recombinant vector, and abiologically pure culture of the recombinant bacterium so transformed.In preferred embodiments, the bacterium preferably being B.thuringiensis such as the recombinant strain EG11402 (deposited on Dec.14, 1994 with the NRRL having Accession No. B-21366) described inExample 8 and the recombinant strain EG11403 (deposited on Dec. 14, 1994with the NRRL having Accession No. B-21367) described in Example 7. Inanother preferred embodiment, the bacterium is preferably E. coli, suchas the recombinant strains EG11460 (deposited on Dec. 14, 1994 with theNRRL having Accession No. B-21364). All strains deposited with the NRRLwere deposited in the Patent Culture Collection under the terms of theBudapest Treaty, and viability statements pursuant to InternationalReceipt Form BP/4 were obtained.

2.1 CryET33 and CryET34 DNA Segments

The present invention also concerns DNA segments, that can be isolatedfrom virtually any source, that are free from total genomic DNA and thatencode the novel peptides disclosed herein. DNA segments encoding thesepeptide species may prove to encode proteins, polypeptides, subunits,functional domains, and the like of crystal protein-related or othernon-related gene products. In addition these DNA segments may besynthesized entirely in vitro using methods that are well-known to thoseof skill in the art.

The cryET33 gene has a nucleotide base sequence shown in FIG. 1A, FIG.1B, and FIG. 1C. The cryET33 gene (SEQ ID NO:1) encodes the 29-kDaCryET33 protein having an amino acid sequence shown in FIG. 1A, FIG. 1B,and FIG. 1C (SEQ ID NO:3). The cryET34 gene (SEQ ID NO:2) encodes the14-kDa CryET34 protein having an amino acid sequence shown in FIG. 1A,FIG. 1B, and FIG. 1C (SEQ ID NO:4).

As used herein, the term “DNA segment” refers to a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species.Therefore, a DNA segment encoding a crystal protein or peptide refers toa DNA segment that contains crystal protein coding sequences yet isisolated away from, or purified free from, total genomic DNA of thespecies from which the DNA segment is obtained, which in the instantcase is the genome of the Gram-positive bacterial genus, Bacillus, andin particular, the species of Bacillus known as B. thuringiensis.Included within the term “DNA segment”, are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phagemids, phage, viruses, and the like.

Similarly, a DNA segment comprising an isolated or purified crystalprotein-encoding gene refers to a DNA segment which may include inaddition to peptide encoding sequences, certain other elements such as,regulatory sequences, isolated substantially away from other naturallyoccurring genes or protein-encoding sequences. In this respect, the term“gene” is used for simplicity to refer to a functional protein-,polypeptide- or peptide-encoding unit. As will be understood by those inthe art, this functional term includes both genomic sequences, operonsequences and smaller engineered gene segments that express, or may beadapted to express, proteins, polypeptides or peptides.

“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a gene encoding a bacterial crystalprotein, forms the significant part of the coding region of the DNAsegment, and that the DNA segment does not contain large portions ofnaturally-occurring coding DNA, such as large chromosomal fragments orother functional genes or operon coding regions. Of course, this refersto the DNA segment as originally isolated, and does not exclude genes,recombinant genes, synthetic linkers, or coding regions later added tothe segment by the hand of man.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences that encode a Crypeptide species that includes within its amino acid sequence an aminoacid sequence essentially as set forth in SEQ ID NO:3 or SEQ ID NO:4.

The term “a sequence essentially as set forth in SEQ ID NO:3 or SEQ IDNO:4,” means that the sequence substantially corresponds to a portion ofthe sequence of either SEQ ID NO:3 or SEQ ID NO:4 and has relatively fewamino acids that are not identical to, or a biologically functionalequivalent of, the amino acids of any of these sequences. The term“biologically functional equivalent” is well understood in the art andis further defined in detail herein (e.g., see IllustrativeEmbodiments). Accordingly, sequences that have between about 70% andabout 80%, or more preferably between about 81% and about 90%, or evenmore preferably between about 91% and about 99% amino acid sequenceidentity or functional equivalence to the amino acids of SEQ ID NO:3 orSEQ ID NO:4 will be sequences that are “essentially as set forth in SEQID NO:3 or SEQ ID NO:4.”

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences that may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared thatinclude a short contiguous stretch encoding either of the peptidesequences disclosed in SEQ ID NO:3 or SEQ ID NO:4, or that are identicalto or complementary to DNA sequences which encode any of the peptidesdisclosed in SEQ ID NO:3 or SEQ ID NO:4, and particularly those DNAsegments disclosed in SEQ ID NO:1 or SEQ ID NO:2. For example, DNAsequences such as about 18 nucleotides, and that are up to about 10,000,about 5,000, about 3,000, about 2,000, about 1,000, about 500, about200, about 100, about 50, and about 14 base pairs in length (includingall intermediate lengths) are also contemplated to be useful.

It will be readily understood that “intermediate lengths”, in thesecontexts, means any length between the quoted ranges, such as 18, 19,20, 21, 22, 23, etc.; 30, 31, 32, etc.; 50, 51, 52, 53, etc.; 100, 101,102, 103, etc.; 150, 151, 152, 153, etc.; including all integers throughthe 200-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; and up toand including sequences of about 5200 nucleotides and the like.

It will also be understood that this invention is not limited to theparticular nucleic acid sequences which encode peptides of the presentinvention, or which encode the amino acid sequences of SEQ ID NO:3 orSEQ ID NO:4, including those DNA sequences which are particularlydisclosed in SEQ ID NO:1 or SEQ ID NO:2. Recombinant vectors andisolated DNA segments may therefore variously include the peptide-codingregions themselves, coding regions bearing selected alterations ormodifications in the basic coding region, or they may encode largerpolypeptides that nevertheless include these peptide-coding regions ormay encode biologically functional equivalent proteins or peptides thathave variant amino acids sequences.

The DNA segments of the present invention encompassbiologically-functional, equivalent peptides. Such sequences may ariseas a consequence of codon degeneracy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally-equivalent proteins orpeptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the protein or to test mutants inorder to examine activity at the molecular level.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the peptide-coding regions are aligned within the same expressionunit with other proteins or peptides having desired functions, such asfor purification or immunodetection purposes (e.g., proteins that may bepurified by affinity chromatography and enzyme label coding regions,respectively).

Recombinant vectors form further aspects of the present invention.Particularly useful vectors are contemplated to be those vectors inwhich the coding portion of the DNA segment, whether encoding a fulllength protein or smaller peptide, is positioned under the control of apromoter. The promoter may be in the form of the promoter that isnaturally associated with a gene encoding peptides of the presentinvention, as may be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR™ technology, in connection with thecompositions disclosed herein.

2.2 CryET33 and CryET34 DNA Segments as Hybridization Probes and Primers

In addition to their use in directing the expression of crystal proteinsor peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least a 14 nucleotidelone contiguous sequence that has the same sequence as, or iscomplementary to, a 14 nucleotide long contiguous DNA segment of SEQ IDNO:1 or SEQ ID NO:2 will find particular utility. Longer contiguousidentical or complementary sequences, e.g., those of about 20, 30, 40,50, 100, 200, 500, 1000, 2000, 5000 bp, etc. (including all intermediatelengths and up to and including the full-length sequence of 5200basepairs will also be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize tocrystal protein-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of 10-14, 15-20, 30, 50, or even of 100-200nucleotides or so, identical or complementary to DNA sequences of SEQ IDNO:1 or SEQ ID NO:2, are particularly contemplated as hybridizationprobes for use in, e.g., Southern and Northern blotting. Smallerfragments will generally find use in hybridization embodiments, whereinthe length of the contiguous complementary region may be varied, such asbetween about 10-14 and about 100 or 200 nucleotides, but largercontiguous complementary stretches may be used, according to the lengthcomplementary sequences one wishes to detect.

Of course, fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as the PCR™ technology of U.S. Pat. Nos. 4,683,195 and4.683,202 (each incorporated herein by reference), by introducingselected sequences into recombinant vectors for recombinant production,and by other recombinant DNA techniques generally known to those ofskill in the art of molecular biology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids. e.g., onewill select relatively low salt and/or high temperature conditions, suchas provided by about 0.02 M to about 0.15 M NaCl at temperatures ofabout 50° C. to about 70° C. Such selective conditions tolerate little,if any, mismatch between the probe and the template or target strand,and would be particularly suitable for isolating crystalprotein-encoding DNA segments. Detection of DNA segments viahybridization is well-known to those of skill in the art, and theteachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (each incorporatedherein by reference) are exemplary of the methods of hybridizationanalyses. Teachings such as those found in the texts of Maloy et al.,1993; Segal 1976; Prokop, 1991; and Kuby, 1991, are particularlyrelevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate crystalprotein-encoding sequences from related species, functional equivalents,or the like, less stringent hybridization conditions will typically beneeded in order to allow formation of the heteroduplex. In thesecircumstances, one may desire to employ conditions such as about 0.15 Mto about 0.9 M salt, at temperatures ranging from about 20° C. to about55° C. Cross-hybridizing species can thereby be readily identified aspositively hybridizing signals with respect to control hybridizations.In any case, it is generally appreciated that conditions can be renderedmore stringent by the addition of increasing amounts of formamide, whichserves to destabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and thus will generally be a method of choice depending on the desiredresults.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmentally undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

2.3 Recombinant Vectors and Crystal Protein Expression

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a crystal protein orpeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anybacterial, viral, eukaryotic, or plant cell. Naturally, it will beimportant to employ a promoter that effectively directs the expressionof the DNA segment in the cell type, organism, or even animal, chosenfor expression. The use of promoter and cell type combinations forprotein expression is generally known to those of skill in the art ofmolecular biology, for example, see Sambrook et al., 1989. The promotersemployed may be constitutive, or inducible, and can be used under theappropriate conditions to direct high level expression of the introducedDNA segment, such as is advantageous in the large-scale production ofrecombinant proteins or peptides. Appropriate promoter systemscontemplated for use in high-level expression include, but are notlimited to, the Pichia expression vector system (Pharmacia LKBBiotechnology).

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of crystal peptidesor epitopic core regions, such as may be used to generate anti-crystalprotein antibodies, also falls within the scope of the invention. DNAsegments that encode peptide antigens from about 8 to about 50 aminoacids in length, or more preferably, from about 8 to about 30 aminoacids in length, or even more preferably, from about 8 to about 20 aminoacids in length are contemplated to be particularly useful. Such peptideepitopes may be amino acid sequences which comprise contiguous aminoacid sequences from SEQ ID NO:3 or SEQ ID NO:4.

2.4 Crystal Protein Transgenes and Transgenic Plants

In yet another aspect, the present invention provides methods forproducing a transgenic plant which expresses a nucleic acid segmentencoding the novel crystal protein of the present invention. The processof producing transgenic plants is well-known in the art. In general, themethod comprises transforming a suitable host cell with a DNA segmentwhich contains a promoter operatively linked to a coding region thatencodes a B. thuringiensis CryET33 or CryET34 crystal protein. Such acoding region is generally operatively linked to atranscription-terminating region, whereby the promoter is capable ofdriving the transcription of the coding region in the cell, and henceproviding the cell the ability to produce the recombinant protein invivo. Alternatively, in instances where it is desirable to control,regulate, or decrease the amount of a particular recombinant crystalprotein expressed in a particular transgenic cell, the invention alsoprovides for the expression of crystal protein antisense mRNA. The useof antisense mRNA as a means of controlling or decreasing the amount ofa given protein of interest in a cell is well-known in the art.

Another aspect of the invention comprises transgenic plants whichexpress a gene or gene segment encoding one or more of the novelpolypeptide compositions disclosed herein. As used herein, the term“transgenic plant” is intended to refer to a plant that has incorporatedDNA sequences, including but not limited to genes which are perhaps notnormally present, DNA sequences not normally transcribed into RNA ortranslated into a protein (“expressed”), or any other genes or DNAsequences which one desires to introduce into the non-transformed plant,such as genes which may normally be present in the non-transformed plantbut which one desires to either genetically engineer or to have alteredexpression.

It is contemplated that in some instances the genome of a transgenicplant of the present invention will have been augmented through thestable introduction of one or more cryET33 or cryET34 transgenes, eithernative, synthetically modified, or mutated. In some instances, more thanone transgene will be incorporated into the genome of the transformedhost plant cell. Such is the case when more than one crystalprotein-encoding DNA segment is incorporated into the genome of such aplant. In certain situations, it may be desirable to have one, two,three, four, or even more B. thuringiensis crystal proteins (eithernative or recombinantly-engineered) incorporated and stably expressed inthe transformed transgenic plant.

A preferred gene which may be introduced includes, for example, acrystal protein-encoding DNA sequence from bacterial origin, andparticularly one or more of those described herein which are obtainedfrom Bacillus spp. Highly preferred nucleic acid sequences are thoseobtained from B. thuringiensis, or any of those sequences which havebeen genetically engineered to decrease or increase the insecticidalactivity of the crystal protein in such a transformed host cell.

Means for transforming a plant cell and the preparation of a transgeniccell line are well-known in the art, and are discussed herein. Vectors,plasmids, cosmids, YACs (yeast artificial chromosomes) and DNA segmentsfor use in transforming such cells will, of course, generally compriseeither the operons, genes, or gene-derived sequences of the presentinvention, either native, or synthetically-derived, and particularlythose encoding the disclosed crystal proteins. These DNA constructs canfurther include structures such as promoters, enhancers, polylinkers, oreven gene sequences which have positively- or negatively-regulatingactivity upon the particular genes of interest as desired. The DNAsegment or gene may encode either a native or modified crystal protein,which will be expressed in the resultant recombinant cells, and/or whichwill impart an improved phenotype to the regenerated plant.

Such transgenic plants may be desirable for increasing the insecticidalresistance of a monocotyledonous or dicotyledonous plant, byincorporating into such a plant, a transgenic DNA segment encoding oneor more CryET33 and/or CryET34 crystal proteins which is toxic toColeopteran insects. Particularly preferred plants include turf grasses,wheat, vegetables, ornamental plants, fruit trees, and the like.

In a related aspect, the present invention also encompasses a seedproduced by the transformed plant, a progeny from such seed, and a seedproduced by the progeny of the original transgenic plant, produced inaccordance with the above process. Such progeny and seeds will have acrystal protein-encoding transgene stably incorporated into theirgenome, and such progeny plants will inherit the traits afforded by theintroduction of a stable transgene in Mendelian fashion. All suchtransgenic plants having incorporated into their genome transgenic DNAsegments encoding one or more CryET33 and/or CryET34 crystal proteins orpolypeptides are aspects of this invention.

2.5 Site-Specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 13 orabout 14 or about 16 or about 17 up to and including about 18 or about19 or about 20, or about 21, 22, 23, 24, 25 26, 27, 28, 29 or even about30, 40, or about 50 or so nucleotides in length is preferred, with about5 to 10 residues on both sides of the junction of the sequence beingaltered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

2.6 CryET33 and CryET34 Antibody Compositions and Methods of Making

In particular embodiments, the inventors contemplate the use ofantibodies, either monoclonal or polyclonal which bind to the crystalproteins disclosed herein. Means for preparing and characterizingantibodies are well known in the art (See, e.g., Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporatedherein by reference). The methods for generating monoclonal antibodies(mAbs) generally begin along the same lines as those for preparingpolyclonal antibodies. Briefly, a polyclonal antibody is prepared byimmunizing an animal with an immunogenic composition in accordance withthe present invention and collecting antisera from that immunizedanimal. A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of anti-antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because ofthe relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified crystal protein, polypeptide or peptide. Theimmunizing composition is administered in a manner effective tostimulate antibody producing cells. Rodents such as mice and rats arepreferred animals, however, the use of rabbit, sheep, or frog cells isalso possible. The use of rats may provide certain advantages (Goding,1986, pp. 60-61), but mice are preferred, with the BALB/c mouse beingmost preferred as this is most routinely used and generally gives ahigher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal, are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83,1984). For example, where the immunized animal is a mouse, one may useP3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11,MPC11X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use R210.RCY3,Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 andUC729-6 are all useful in connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, (Gefter et al., 1977). The use of electrically inducedfusion methods is also appropriate (Goding, 1986, pp. 71-74).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may beexplorited for mAb production in two basic ways. A sample of thehybridoma can be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion. The injected animal developstumors secreting the specific monoclonal antibody produced by the fusedcell hybrid. The body fluids of the animal, such as serum or ascitesfluid, can then be tapped to provide mAbs in high concentration. Theindividual cell lines could also be cultured in vitro, where the mAbsare naturally secreted into the culture medium from which they can bereadily obtained in high concentrations. mAbs produced by either meansmay be further purified, if desired, using filtration, centrifugationand various chromatographic methods such as HPLC or affinitychromatography.

2.7 Elisas and Immunoprecipitation

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating crystal protein antigen sequences areimmobilized onto a selected surface, preferably a surface exhibiting aprotein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of milk powder. Thisallows for blocking of nonspecific adsorption sites on the immobilizingsurface and thus reduces the background caused by nonspecific binding ofantisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hours, at temperatures preferably on theorder of about 25° to about 27° C. Following incubation, theantisera-contacted surface is washed so as to remove non-immunocomplexedmaterial. A preferred washing procedure includes washing with a solutionsuch as PBS/Tween®, or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease orperoxidase-conjugated anti-human IgG for a period of time and underconditions which favor the development of immunocomplex formation (e.g.,incubation for 2 hours at room temperature in a PBS-containing solutionsuch as PBS Tween®).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case or peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

The anti-crystal protein antibodies of the present invention areparticularly useful for the isolation of other crystal protein antigensby immunoprecipitation. Immunoprecipitation involves the separation ofthe target antigen component from a complex mixture, and is used todiscriminate or isolate minute amounts of protein. For the isolation ofmembrane proteins cells must be solubilized into detergent micelles.Nonionic salts are preferred, since other agents such as bile salts,precipitate at acid pH or in the presence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.enzyme-substrate pairs.

2.8 Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-peptide antibodies may beused as high-affinity primary reagents for the identification ofproteins immobilized onto a solid support matrix, such asnitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

2.9 Crystal Protein Screening and Detection Kits

The present invention contemplates methods and kits for screeningsamples suspected of containing crystal protein polypeptides or crystalprotein-related polypeptides, or cells producing such polypeptides. Akit may contain one or more antibodies of the present invention, and mayalso contain reagent(s) for detecting an interaction between a sampleand an antibody of the present invention. The provided reagent(s) can beradio-, fluorescently- or enzymatically-labeled. The kit can contain aknown radiolabeled agent capable of binding or interacting with anucleic acid or antibody of the present invention.

The reagent(s) of the kit can be provided as a liquid solution, attachedto a solid support or as a dried powder. Preferably, when the reagent(s)are provided in a liquid solution, the liquid solution is an aqueoussolution. Preferably, when the reagent(s) provided are attached to asolid support, the solid support can be chromatograph media, a testplate having a plurality of wells, or a microscope slide. When thereagent(s) provided are a dry powder, the powder can be reconstituted bythe addition of a suitable solvent, that may be provided.

In still further embodiments, the present invention concernsimmunodetection methods and associated kits. It is proposed that thecrystal proteins or peptides of the present invention may be employed todetect antibodies having reactivity therewith, or, alternatively,antibodies prepared in accordance with the present invention, may beemployed to detect crystal proteins or crystal protein-relatedepitope-containing peptides. In general, these methods will includefirst obtaining a sample suspected of containing such a protein, peptideor antibody, contacting the sample with an antibody or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of an immunocomplex, andthen detecting the presence of the immunocomplex.

In general, the detection of immunocomplex formation is quite well knownin the art and may be achieved through the application of numerousapproaches. For example, the present invention contemplates theapplication of ELISA, RIA, immunoblot (e.g., dot blot), indirectimmunofluorescence techniques and the like. Generally, immunocomplexformation will be detected through the use of a label, such as aradiolabel or an enzyme tag (such as alkaline phosphatase, horseradishperoxidase, or the like). Of course, one may find additional advantagesthrough the use of a secondary binding ligand such as a second antibodyor a biotin/avidin ligand binding arrangement, as is known in the art.

For assaying purposes, it is proposed that virtually any samplesuspected of comprising either a crystal protein or peptide or a crystalprotein-related peptide or antibody sought to be detected, as the casemay be, may be employed. It is contemplated that such embodiments mayhave application in the titering of antigen or antibody samples, in theselection of hybridomas, and the like. In related embodiments, thepresent invention contemplates the preparation of kits that may beemployed to detect the presence of crystal proteins or related peptidesand/or antibodies in a sample. Samples may include cells, cellsupernatants, cell suspensions, cell extracts, enzyme fractions, proteinextracts, or other cell-free compositions suspected of containingcrystal proteins or peptides. Generally speaking, kits in accordancewith the present invention will include a suitable crystal protein,peptide or an antibody directed against such a protein or peptide,together with an immunodetection reagent and a means for containing theantibody or antigen and reagent. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, as noted above, a number of exemplarylabels are known in the art and all such labels may be employed inconnection with the present invention.

The container will generally include a vial into which the antibody,antigen or detection reagent may be placed, and preferably suitablyaliquotted. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and reagentcontainers in close confinement for commercial sale. Such containers mayinclude injection or blow-molded plastic containers into which thedesired vials are retained.

2.10 Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions, free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies. In particular, the invention concerns epitopic coresequences derived from Cry proteins or peptides.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-crystal proteinantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a crystal protein or polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the crystal protein orpolypeptide will also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of Cry immunodominant epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1989; Wolf et al., 1988: U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 8 to about 20 amino acids in length,and more preferably about 8 to about 15 amino acids in length. It isproposed that shorter antigenic crystal protein-derived peptides willprovide advantages in certain circumstances, for example, in thepreparation of immunologic detection assays. Exemplary advantagesinclude the ease of preparation and purification, the relatively lowcost and improved reproducibility of production, and advantageousbiodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to crystal proteins,and in particular Cry and Cry-related sequences. These epitopic coresequences are identified herein in particular aspects as hydrophilicregions of the particular polypeptide antigen. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation, and, hence, elicit specific antibody production.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on the crystal protein-directed antibodiesdisclosed herein. Additionally or alternatively, an epitopic coresequence is one that will elicit antibodies that are cross-reactive withantibodies directed against the peptide compositions of the presentinvention. It will be understood that in the context of the presentdisclosure, the term “complementary” refers to amino acids or peptidesthat exhibit an attractive force towards each other. Thus, certainepitope core sequences of the present invention may be operationallydefined in terms of their ability to compete with or perhaps displacethe binding of the desired protein antigen with the correspondingprotein-directed antisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence expected by the present disclosure would generally be on theorder of about 8 amino acids in length, with sequences on the order of10 to 20 being more preferred. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison, Wis.) mayalso be useful in designing synthetic peptides in accordance with thepresent disclosure.

Syntheses of epitopic sequences, or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquotted in predetermined amounts andstored in conventional manners, such as in aqueous solutions or, evenmore preferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat about 4° C., or more preferably, frozen. Of course, where thepeptides are stored in a lyophilized or powdered state, they may bestored virtually indefinitely, e.g., in metered aliquots that may berehydrated with a predetermined amount of water (preferably distilled)or buffer prior to use.

2.11 Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. In particular embodiments ofthe invention, mutated crystal proteins are contemplated to be usefulfor increasing the insecticidal activity of the protein, andconsequently increasing the insecticidal activity and/or expression ofthe recombinant transgene in a plant cell. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thecodons given in Table 2.

TABLE 2 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic Asp D GAC GAU acid Glutamic Glu E GAA GAG acidPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101. incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

2.12 Crystal Protein Compositions as Insecticides and Methods of Use

The inventors contemplate that the crystal protein compositionsdisclosed herein will find particular utility as insecticides fortopical and/or systemic application to field crops, grasses, fruits andvegetables, and ornamental plants. In a preferred embodiment, thebioinsecticide composition comprises an oil flowable suspension ofbacterial cells which expresses a novel crystal protein disclosedherein. Preferably the cells are B. thuringiensis EG10327 cells,however, any such bacterial host cell expressing the novel nucleic acidsegments disclosed herein and producing a crystal protein iscontemplated to be useful, such as B. thuringiensis, B. megaterium, B.subtilis, E. coli, or Pseudonmonas spp.

In another important embodiment, the bioinsecticide compositioncomprises a water dispersible granule. This granule comprises bacterialcells which expresses a novel crystal protein disclosed herein.Preferred bacterial cells are B. thuringiensis EG10327 cells, however,bacteria such as B. thuringiensis, B. megaterium, B. subtilis, E. coli,or Pseudomonas spp. cells transformed with a DNA segment disclosedherein and expressing the crystal protein are also contemplated to beuseful.

In a third important embodiment, the bioinsecticide compositioncomprises a wettable powder, dust, pellet, or collodial concentrate.This powder comprises bacterial cells which expresses a novel crystalprotein disclosed herein. Preferred bacterial cells are B. thuringiensisEG10327 cells, however, bacteria such as B. thuringiensis, B.megaterium, B. subtilis, E. coli, or Pseudomonas spp. cells transformedwith a DNA segment disclosed herein and expressing the crystal proteinare also contemplated to be useful. Such dry forms of the insecticidalcompositions may be formulated to dissolve immediately upon wetting, oralternatively, dissolve in a controlled-release, sustained-release, orother time-dependent manner.

In a fourth important embodiment, the bioinsecticide compositioncomprises an aqueous suspension of bacterial cells such as thosedescribed above which express the crystal protein. Such aqueoussuspensions may be provided as a concentrated stock solution which isdiluted prior to application, or alternatively, as a diluted solutionready-to-apply.

For these methods involving application of bacterial cells, the cellularhost containing the crystal protein gene(s) may be grown in anyconvenient nutrient medium, where the DNA construct provides a selectiveadvantage, providing for a selective medium so that substantially all orall of the cells retain the B. thuringiensis gene. These cells may thenbe harvested in accordance with conventional ways. Alternatively, thecells can be treated prior to harvesting.

When the insecticidal compositions comprise intact B. thuringiensiscells expressing the protein of interest, such bacteria may beformulated in a variety of ways. They may be employed as wettablepowders, granules or dusts, by mixing with various inert materials, suchas inorganic minerals (phyllosilicates, carbonates, sulfates,phosphates, and the like) or botanical materials (powdered corncobs,rice hulls, walnut shells, and the like). The formulations may includespreader-sticker adjuvants, stabilizing agents, other pesticidaladditives, or surfactants. Liquid formulations may be aqueous-based ornon-aqueous and employed as foams, suspensions, emulsifiableconcentrates, or the like. The ingredients may include rheologicalagents, surfactants, emulsifiers, dispersants, or polymers.

Alternatively, the novel CryET33 and/or CryET34 proteins may be preparedby native or recombinant bacterial expression systems in vitro andisolated for subsequent field application. Such protein may be either incrude cell lysates, suspensions, colloids, etc., or alternatively may bepurified, refined, buffered, and/or further processed, beforeformulating in an active biocidal formulation. Likewise, under certaincircumstances, it may be desirable to isolate crystals and/or sporesfrom bacterial cultures expressing the crystal protein and applysolutions, suspensions, or collodial preparations of such crystalsand/or spores as the active bioinsecticidal composition.

Regardless of the method of application, the amount of the activecomponent(s) is applied at an insecticidally-effective amount, whichwill vary depending on such factors as, for example, the specificcoleopteran insects to be controlled, the specific plant or crop to betreated, the environmental conditions, and the method, rate, andquantity of application of the insecticidally-active composition.

The insecticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, dessicated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in or oil (vegetable or mineral), or water oror oil/water emulsions, or as a wettable powder, or in combination withany other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, e.g., inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in insecticide formulationtechnology; these are well known to those skilled in insecticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the insecticidal composition withsuitable adjuvants using conventional formulation techniques.

The insecticidal compositions of this invention are applied to theenvironment of the target coleopteran insect, typically onto the foliageof the plant or crop to be protected, by conventional methods,preferably by spraying. The strength and duration of insecticidalapplication will be set with regard to conditions specific to theparticular pest(s), crop(s) to be treated and particular environmentalconditions. The proportional ratio of active ingredient to carrier willnaturally depend on the chemical nature, solubility, and stability ofthe insecticidal composition, as well as the particular formulationcontemplated.

Other application techniques, e.g., dusting, sprinkling, soaking, soilinjection, seed coating, seedling coating, spraying, aerating, misting,atomizing, and the like, are also feasible and may be required undercertain circumstances such as e.g., insects that cause root or stalkinfestation, or for application to delicate vegetation or ornamentalplants. These application procedures are also well-known to those ofskill in the art.

The insecticidal composition of the invention may be employed in themethod of the invention singly or in combination with other compounds,including and not limited to other pesticides. The method of theinvention may also be used in conjunction with other treatments such assurfactants, detergents, polymers or time-release formulations. Theinsecticidal compositions of the present invention may be formulated foreither systemic or topical use.

The concentration of insecticidal composition which is used forenvironmental, systemic, or foliar application will vary widelydepending upon the nature of the particular formulation, means ofapplication, environmental conditions, and degree of biocidal activity.Typically, the bioinsecticidal composition will be present in theapplied formulation at a concentration of at least about 1% by weightand may be up to and including about 99% by weight. Dry formulations ofthe compositions may be from about 1% to about 99% or more by weight ofthe composition, while liquid formulations may generally comprise fromabout 1% to about 99% or more of the active ingredient by weight.Formulations which comprise intact bacterial cells will generallycontain from about 10⁴ to about 10⁷ cells/mg.

The insecticidal formulation may be administered to a particular plantor target area in one or more applications as needed, with a typicalfield application rate per hectare ranging on the order of from about 50g to about 500 g of active ingredient, or of from about 500 g to about1000 g, or of from about 1000 g to about 5000 g or more of activeingredient.

3. BRIEF DESCRIPTION OF THE DRAWINGS

The drawings form part of the present specification and are included tofurther demonstrate certain aspects of the present invention. Theinvention may be better understood by reference to one or more of thesedrawings in combination with the detailed description of specificembodiments presented herein.

FIG. 1A, FIG. 1B, and FIG. 1C show the nucleotide base sequence of thecryET33 gene (SEQ ID NO:1) and the cryET34 gene (SEQ ID NO:2), and thededuced amino acid sequence of the CryET33 protein (SEQ ID NO:3) and theCryET34 protein (SEQ ID NO:4).

FIG. 2 shows a restriction map of pEG246. The locations and orientationsof the cryET33 gene (SEQ ID NO:1) and the cryE734 gene (SEQ ID NO:2) areindicated by arrows. pEG246 is functional in E. coli since it is derivedfrom pBR322, and is ampicillin resistant (Amp^(R)). The abbreviationsfor the restriction endonuclease cleavage sites are as follows: R=EcoRI,B=BamHI. Also shown in FIG. 2 is a one kilobase (1 kb) size marker.

FIG. 3, aligned with and based on the same scale as FIG. 2, shows arestriction map of pEG1246. The locations and orientations of thecryET33 gene (SEQ ID NO:1) and the cryET34 gene (SEQ ID NO:2) areindicated by arrows. pEG1246 is derived from plasmid pEG246 (FIG. 2) andcontains the Bacillus spp. plasmid, pNN101 (which expresses bothchloramphenicol resistance [Cam^(R)] and tetracycline resistance[Tet^(R)]) inserted into the BamHI site of pEG246. pEG1246 is functionalin both E. coli and B. thuringiensis. Abbreviations are the same asthose for FIG. 2.

4. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

4.1 Some Advantages of the Invention

B. thuringiensis EG10327 is a naturally-occurring strain that exhibitsinsecticidal activity against coleopteran insects including boll weevil,red flour beetle larvae (Tribolium castaneum) and Japanese beetle larvae(Popillia japonica). B. thuringiensis EG2158 contains colepteran-toxiccrystal protein genes similar to, or identical with, the crystal proteingenes of EG10327. Two novel crystal toxin genes, designated cryET33 andcryvET34, were cloned from EG2158. The cryET33gene encodes the 29-kDaCryET33 crystal protein, and the cryET34 gene encodes the 14-kDa CryET34crystal protein. The CryET33 and CryET34 crystal proteins are toxic tored flour beetle larvae, boll weevil larvae, and Japanese beetle larvae.

4.2 Definitions

The following words and phrases have the meanings set forth below.

Expression: The combination of intracellular processes, includingtranscription and translation undergone by a coding DNA molecule such asa structural gene to produce a polypeptide.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a structural gene and towhich RNA polymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Regeneration: The process of growing a plant from a plant cell (e.g.,plant protoplast or explant).

Structural gene: A gene that is expressed to produce a polypeptide.

Transformation: A process of introducing an exogenous DNA sequence(e.g., a vector, a recombinant DNA molecule) into a cell or protoplastin which that exogenous DNA is incorporated into a chromosome or iscapable of autonomous replication.

Transformed cell: A cell whose DNA has been altered by the introductionof an exogenous DNA molecule into that cell.

Transgenic cell: Any cell derived or regenerated from a transformed cellor derived from a transgenic cell. Exemplary transgenic cells includeplant calli derived from a transformed plant cell and particular cellssuch as leaf, root, stem, e.g., somatic cells, or reproductive (germ)cells obtained from a transgenic plant.

Transgenic plant: A plant or progeny thereof derived from a transformedplant cell or protoplast, wherein the plant DNA contains an introducedexogenous DNA molecule not originally present in a native,non-transgenic plant of the same strain. The terms “transgenic plant”and “transformed plant” have sometimes been used in the art assynonymous terms to define a plant whose DNA contains an exogenous DNAmolecule. However, it is thought more scientifically correct to refer toa regenerated plant or callus obtained from a transformed plant cell orprotoplast as being a transgenic plant, and that usage will be followedherein.

Vector: A DNA molecule capable of replication in a host cell and/or towhich another DNA segment can be operatively linked so as to bring aboutreplication of the attached segment. A plasmid is an exemplary vector.

4.3 Probes and Primers

In another aspect, DNA sequence information provided by the inventionallows for the preparation of relatively short DNA (or RNA) sequenceshaving the ability to specifically hybridize to gene sequences of theselected polynucleotides disclosed herein. In these aspects, nucleicacid probes of an appropriate length are prepared based on aconsideration of a selected crystal protein gene sequence, e.g., asequence such as that shown in SEQ ID NO:1or SEQ ID NO:2. The ability ofsuch DNAs and nucleic acid probes to specifically hybridize to a crystalprotein-encoding gene sequence lends them particular utility in avariety of embodiments. Most importantly, the probes may be used in avariety of assays for detecting the presence of complementary sequencesin a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingPCR™ technology. Segments of related crystal protein genes from otherspecies may also be amplified by PCR™ using such primers.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes sequences that are complementary to at leasta 14 to 30 or so long nucleotide stretch of a crystal protein-encodingsequence, such as that shown in SEQ ID NO:1 or SEQ ID NO:2. A size of atleast 14 nucleotides in length helps to ensure that the fragment will beof sufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 14 bases in length are generally preferred, though, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained.One will generally prefer to design nucleic acid molecules havinggene-complementary stretches of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. Nos. 4,683,195, and 4,683,202, herein incorporated byreference, or by excising selected DNA fragments from recombinantplasmids containing appropriate inserts and suitable restriction sites.

4.4 Expression Vectors

The present invention contemplates an expression vector comprising apolynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to an coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region.

As used herein, the term “operatively linked” means that a promoter isconnected to an coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region are well known inthe art.

In a preferred embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is preferable in aBacillus host cell. Preferred host cells include B. thuringiensis, B.megaterium, B. Subtilis, and related bacilli, with B. thuringiensis hostcells being highly preferred. Promoters that function in bacteria arewell-known in the art. An exemplary and preferred promoter for theBacillus crystal proteins include any of the known crystal protein genepromoters, including the cryET33 and cryET34 gene promoters.Alternatively, mutagenized or recombinant crystal protein-encoding genepromoters may be engineered by the hand of man and used to promoteexpression of the novel gene segments disclosed herein.

In an alternate embodiment, the recombinant expression of DNAs encodingthe crystal proteins of the present invention is performed using atransformed Gram-negative bacterium such as an E. coli or Pseudomonasspp. host cell. Promoters which function in high-level expression oftarget polypeptides in E. coli and other Gram-negative host cells arealso well-known in the art.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in plants. Promoters that function in plants are also wellknown in the art. Useful in expressing the polypeptide in plants arepromoters that are inducible, viral, synthetic, constitutive asdescribed (Poszkowski et al., 1989; Odell et al., 1985), and temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989).

A promoter is also selected for its ability to direct the transformedplant cell's or transgenic plant's transcriptional activity to thecoding region. Structural genes can be driven by a variety of promotersin plant tissues. Promoters can be near-constitutive, such as the CaMV35S promoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots.

Where the promoter is a near-constitutive promoter such as CaMV 35S,increases in polypeptide expression are found in a variety oftransformed plant tissues (e.g., callus, leaf, seed and root).Alternatively, the effects of transformation can be directed to specificplant tissues by using plant integrating vectors containing atissue-specific promoter.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The Lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983 t Lindstrom et al., 1990.)

An expression vector containing a coding region that encodes apolypeptide of interest is engineered to be under control of the lectinpromoter and that vector is introduced into plants using, for example, aprotoplast transformation method (Dhir et al., 1991). The expression ofthe polypeptide is directed specifically to the seeds of the transgenicplant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Exemplary tissue-specific promoters are corn sucrose synthetase 1 (Yanget al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989), cornlight harvesting complex (Simpson, 1986), corn heat shock protein (Odellet al., 1985), pea small subunit RuBP carboxylase (Poulsen et al., 1986;Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), CaMV 35s transcript (Odell et al., 1985) andPotato patatin (Wenzler et al., 1989). Preferred promoters are thecauliflower mosaic virus (CaMV 35S) promoter and the S-E9 small subunitRuBP carboxylase promoter.

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described (Rogers et al.,1987). However, several other plant integrating vector systems are knownto function in plants including pCaMVCN transfer control vectordescribed (Fromm et al., 1985). Plasmid pCaMVCN (available fromPharmacia, Piscataway, N.J.) includes the cauliflower mosaic virus CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e, the chimeric gene containing the nopaline synthasepromoters Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

RNA polymerase transcribes a coding DNA sequence through a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Means for preparing expression vectors are well known in the art.Expression (transformation vectors) used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011, the disclosures of which areincorporated herein by reference. Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A variety of methods has been developed to operatively link DNA tovectors via complementary cohesive termini or blunt ends. For instance,complementary homopolymer tracts can be added to the DNA segment to beinserted and to the vector DNA. The vector and DNA segment are thenjoined by hydrogen bonding between the complementary homopolymeric tailsto form recombinant DNA molecules.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a CryET33 or CryET34 B.thuringiensis crystal protein-encoding gene. In preferred embodiments,such a polypeptide has the amino acid residue sequence of SEQ ID NO:3 orSEQ ID NO:4, or a functional equivalent of those sequences. Inaccordance with such embodiments, a coding region comprising the DNAsequence of SEQ ID NO:1 or the DNA sequence of SEQ ID NO:2 is alsopreferred.

4.5 Characteristics of the Novel Crystal Proteins

The present invention provides novel polypeptides that define a whole ora portion of a B. thuringiensis CryET33 or CryET34 crystal protein.

In a preferred embodiment, the invention discloses and claims anisolated and purified CryET33 protein. The CryET33 protein comprises a267-amino acid sequence, and has a calculated molecular mass of 29,216Da. CryET33 has a calculated isoelectric constant (pI) equal to 4.78.The amino acid composition of the CryET33 protein is given in Table 3.

TABLE 3 AMINO ACID COMPOSITION OF CRYET33 % Amino Acid # Residues TotalAmino Acid # Residues % Total Ala 14 (5.2) Leu 12 (4.5) Arg 5 (1.9) Lys14 (5.2) Asn 22 (8.2) Met 3 (1.1) Asp 12 (4.5) Phe 11 (4.1) Cys 2 (0.7)Pro 12 (4.5) Gln 7 (2.6) Ser 22 (8.2) Glu 15 (5.6) Thr 39 (14.5) Gly 18(6.7) Trp 2 (0.7) His 3 (1.1) Tyr 14 (5.2) Ile 17 (6.3) Val 23 (8.6)Acidic (Asp + Glu) 27 (10.00) Basic (Arg + Lys) 19 (7.1) Aromatic (Phe +Trp + Tyr) 27 (10.0) Hydrophobic (Aromatic + Ile + Leu + Met + 82 (30.5)Val)

In another embodiment, the invention discloses and claims an isolatedand purified CryET34 protein. The CryET34 protein comprises a 126-aminoacid sequence and has a calculated molecular mass of 14,182 Da. Thecalculated isoelectric point (pI) of CryET34 is 4.26. The amino acidcomposition of the CryET34 protein is given in Table 4.

TABLE 4 AMINO ACID COMPOSITION OF CRYET34 % Amino Acid #Residues TotalAmino Acid #Residues % Total Ala 5 (3.9) Leu 4 (3.1) Arg 2 (1.6) Lys 8(6.3) Asn 6 (4.7) Met 2 (1.6) Asp 11 (8.7) Phe 4 (3.1) Cys 2 (1.6) Pro 8(6.3) Gln 4 (3.1) Ser 9 (7.1) Glu 7 (5.5) Thr 13 (10.2) Gly 11 (8.7) Trp3 (2.4) His 1 (0.8) Tyr 11 (8.7) Ile 8 (6.3) Val 7 (5.5) Acidic (Asp +Glu) 18 (14.2) Basic (Arg + Lys) 10 (7.9) Aromatic (Phe + Trp + Tyr) 18(14.2) Hydrophobic (Aromatic + Ile + Leu + Met + 39 (30.7) Val)

4.6 Nomenclature of the Novel Proteins

The inventors have arbitrarily assigned the designations Cry33 andCryET34 to the novel proteins of the invention. Likewise, the arbitrarydesignations of cryET33 and cyET34 have been assigned to the novelnucleic acid sequences which encode these polypeptides, respectively.Formal assignment of gene and protein designations based on the revisednomenclature of crystal protein endotoxins (Table 1) will be assigned bya committee on the nomenclature of B. thuringiensis , formed tosystematically classify B. thuringiensis crystal proteins. The inventorscontemplate that the arbitrarily assigned designations of the presentinvention will be superseded by the official nomenclature assigned tothese sequences.

4.7 Transformed Host Cells and Transgenic Plants

Methods and compositions for transforming a bacterium, a yeast cell, aplant cell, or an entire plant with one or more expression vectorscomprising a crystal protein-encoding gene segment are further aspectsof this disclosure. A transgenic bacterium, yeast cell, plant cell orplant derived from such a transformation process or the progeny andseeds from such a transgenic plant are also further embodiments of theinvention.

Means for transforming bacteria and yeast cells are well known in theart. Typically, means of transformation are similar to those well knownmeans used to transform other bacteria or yeast such as E. coli orSaccharomyces cerevisiae. Methods for DNA transformation of plant cellsinclude Agrobacterium-mediated plant transformation, protoplasttransformation, gene transfer into pollen, injection into reproductiveorgans, injection into immature embryos and particle bombardment. Eachof these methods has distinct advantages and disadvantages. Thus, oneparticular method of introducing genes into a particular plant strainmay not necessarily be the most effective for another plant strain, butit is well known which methods are useful for a particular plant strain.

There are many methods for introducing transforming DNA segments intocells, but not all are suitable for delivering DNA to plant cells.Suitable methods are believed to include virtually any method by whichDNA can be introduced into a cell, such as by Agrobacterium infection,direct delivery of DNA such as, for example, by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993), bydesiccation/inhibition-mediated DNA uptake, by electroporation, byagitation with silicon carbide fibers, by acceleration of DNA coatedparticles, etc. In certain embodiments, acceleration methods arepreferred and include, for example, microprojectile bombardment and thelike.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973;Zatloukal et al., 1992); (2) physical methods such as microinjection(Capecchi, 1980), electroporation (Wong and Neumann, 1982; Fromm et al.,1985; U.S. Pat. No. 5,384,253) and the gene gun (Johnston and Tang,1994; Fynan et al., 1993); (3) viral vectors (Clapp, 1993; Lu et al.,1993; Eglitis and Anderson, 1988a; 1988b); and (4) receptor-mediatedmechanisms (Curie et al., 1991; 1992; Wagner et al., 1992).

4.7.1 Electroporation

The application of brief, high-voltage electric pulses to a variety ofanimal and plant cells leads to the formation of nanometer-sized poresin the plasma membrane. DNA is taken directly into the cell cytoplasmeither through these pores or as a consequence of the redistribution ofmembrane components that accompanies closure of the pores.Electroporation can be extremely efficient and can be used both fortransient expression of clones genes and for establishment of cell linesthat carry integrated copies of the gene of interest. Electroporation,in contrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of the foreign DNA.

The introduction of DNA by means of electroporation, is well-known tothose of skill in the art. In this method, certain cell wall-degradingenzymes, such as pectin-degrading enzymes, are employed to render thetarget recipient cells more susceptible to transformation byelectroporation than untreated cells. Alternatively, recipient cells aremade more susceptible to transformation, by mechanical wounding. Toeffect transformation by electroporation one may employ either friabletissues such as a suspension culture of cells, or embryogenic callus, oralternatively, one may transform immature embryos or other organizedtissues directly. One would partially degrade the cell walls of thechosen cells by exposing them to pectin-degrading enzymes (pectolyases)or mechanically wounding in a controlled manner. Such cells would thenbe recipient to DNA transfer by electroporation, which may be carriedout at this stage, and transformed cells then identified by a suitableselection or screening protocol dependent on the nature of the newlyincorporated DNA.

4.7.2 Microprojectile Bombardment

A further advantageous method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesmay be coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, gold, platinum, and the like.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into maize cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen onto a filter surface covered with corncells cultured in suspension. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectiles aggregate andmay contribute to a higher frequency of transformation by reducingdamage inflicted on the recipient cells by projectiles that are toolarge.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the prebombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

Accordingly. it is contemplated that one may wish to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example. the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

4.7.3 Agrobacterium-Mediated Transfer

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). Further, the integration of the Ti-DNAis a relatively precise process resulting in few rearrangements. Theregion of DNA to be transferred is defined by the border sequences, andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., 1986; Jorgensen et al., 1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant strains whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Therefore, commercially important cereal grains such asrice, corn, and wheat must usually be transformed using alternativemethods. However, as mentioned above, the transformation of asparagususing Agrobacterium can also be achieved (see, for example, Bytebier etal., 1987).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

More preferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for enhanced carboxylase activity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

It is to be understood that two different transgenic plants can also bemated to produce offspring that contain two independently segregatingadded, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated.

4.7.4 Other Transformation Methods

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see e.g.,Potrykus et al., 1985; Lorz et al., 1985: Fromm et al., 1986; Uchimiyaet al., 1986, Callis et al., 1987; Marcotte et al., 1988).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., 1985; Toriyama et al., 1986, Yamada etal., 1986; Abdullah et al., 1986).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, 1988). Inaddition, “particle gun” or high-velocity microprojectile technology canbe utilized. (Vasil, 1992).

Using that latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., 1987; Klein et al., 1988; McCabe et al., 1988). The metalparticles penetrate through several layers of cells and thus allow thetransformation of cells within tissue explants.

4.8 Methods for Producing Insect-Resistant Transgenic Plants

By transforming a suitable host cell, such as a plant cell, with arecombinant cryET33 and/or cryET34 gene-containing segment, theexpression of the encoded crystal protein (i.e., a bacterial crystalprotein or polypeptide having insecticidal activity againstcoleopterans) can result in the formation of insect-resistant plants.

By way of example, one may utilize an expression vector containing acoding region for a B. thuringiensis crystal protein and an appropriateselectable marker to transform a suspension of embryonic plant cells,such as wheat or corn cells using a method such as particle bombardment(Maddock et al., 1991; Vasil et al., 1992) to deliver the DNA coated onmicroprojectiles into the recipient cells. Transgenic plants are thenregenerated from transformed embryonic calli that express theinsecticidal proteins.

The formation of transgenic plants may also be accomplished using othermethods of cell transformation which are known in the art such asAgrobacterium-mediated DNA transfer (Fraley et al., 1983).Alternatively, DNA can be introduced into plants by direct DNA transferinto pollen (Zhou et al., 1983; Hess, 1987; Luo et al., 1988), byinjection of the DNA into reproductive organs of a plant (Pena et al.,1987), or by direct injection of DNA into the cells of immature embryosfollowed by the rehydration of desiccated embryos (Neuhaus et al., 1987;Benbrook et al., 1986).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, 1988). This regenerationand growth process typically includes the steps of selection oftransformed cells, culturing those individualized cells through theusual stages of embryonic development through the rooted plantlet stage.Transgenic embryos and seeds are similarly regenerated. The resultingtransgenic rooted shoots are thereafter planted in an appropriate plantgrowth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983).

This procedure typically produces shoots within two to four months andthose shoots are then transferred to an appropriate root-inducing mediumcontaining the selective agent and an antibiotic to prevent bacterialgrowth. Shoots that rooted in the presence of the selective agent toform plantlets are then transplanted to soil or other media to allow theproduction of roots. These procedures vary depending upon the particularplant strain employed, such variations being well known in the art.

Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants, as discussed before. Otherwise, pollenobtained from the regenerated plants is crossed to seed-grown plants ofagronomically important, preferably inbred lines. Conversely, pollenfrom plants of those important lines is used to pollinate regeneratedplants. A transgenic plant of the present invention containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

A transgenic plant of this invention thus has an increased amount of acoding region (e.g., a cry gene) that encodes the Cry polypeptide ofinterest. A preferred transgenic plant is an independent segregant andcan transmit that gene and its activity to its progeny. A more preferredtransgenic plant is homozygous for that gene, and transmits that gene toall of its offspring on sexual mating. Seed from a transgenic plant maybe grown in the field or greenhouse, and resulting sexually maturetransgenic plants are self-pollinated to generate true breeding plants.The progeny from these plants become true breeding lines that areevaluated for, by way of example, increased insecticidal capacityagainst coleopteran insects, preferably in the field, under a range ofenvironmental conditions. The inventors contemplate that the presentinvention will find particular utility in the creation of transgenicplants of commercial interest including various turf grasses. wheat,corn, rice, barley, oats, a variety of ornamental plants and vegetables,as well as a number of nut- and fruit-bearing trees and plants.

5. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

5.1 Example 1

Isolation of B. thuringiensis EG10327

Crop dust samples were obtained from various sources throughout the U.S.and abroad, typically grain storage facilities. The crop dust sampleswere treated and spread on agar plates to isolate individualBacillus-type colonies as described in U.S. Pat. No. 5,264,364.

The cloned cryIIIA gene, formerly known as the cryC gene of B.thuringiensis strain EG2158, described in Donovan et al., (1988), andthe cloned cryIIIB2 gene, formerly known as the cryIIIC gene of B.thuringiensis strain EG4961, described in Donovan et al., 1992, wereused as probes in colony hybridization procedures. The cryIIIA geneprobe consisted of a radioactively labeled 2.0 kb HindIII-XbaI DNArestriction fragment as described in Donovan et al., 1988. The cryIIIB2gene probe consisted of a radioactively labeled 2.4 kb SspI DNArestriction fragment as described in Donovan et al., 1992. The colonyhybridization procedures were performed as described in U.S. Pat. No.5,264,364.

Approximately 43,000 Bacillus-type colonies from fifty-four crop dustsamples from various locations were probed with theradioactively-labeled cryIIIA and cryIIIB2 probes. One crop dust samplefrom Greece contained approximately 100 naturally-occurringBacillus-type colonies that hybridized with the cryIIIA and cryIIIB2probes. Analysis of several of these naturally-occurring, wild-typecolonies indicated that they were identical B. thuringiensis colonies,and one colony, designated EG10327, was selected for further study. B.thuringiensis strain EG10327 was deposited on Dec. 14, 1994 under theterms of the Budapest Treaty with the NRRL under Accession No. NRRLB-21365.

Subsequently approximately 84,000 Bacillus-type colonies from 105 cropdust samples from various locations were also screened with theradioactively-labeled cryIIIA and cryIIIB2 probes, but without successin identifying any other strains containing novel cryIII-type genes.

B. thuringiensis strain EG10327 was found to be insecticidally-activeagainst the larvae of coleopteran insects, notably, the red flourbeetle, the boll weevil, and the Japanese beetle. Strain EG10327 did nothave measurable insecticidal activity with respect to the southern cornrootworm or the Colorado potato beetle under the assay conditions used.A gene, designated “cryIIIA-truncated”, was isolated from strainEG10327, and its nucleotide base sequence determined. ThecryIIIA-truncated gene was found to be identical with the firsttwo-thirds of the cryIIIA gene (described as the cryC gene in Donovan etal., 1988) but did not contain the final one-third of the cryIIIA gene.The truncated cryIIIA gene of strain EG10327 produced very little, ifany, insecticidal protein and was not further characterized.

5.2 Example 2

Evaluation of the Flagellar Serotype of EG10327

To characterize strain EG10327 several studies were conducted. One studywas performed to characterize its flagellar serotype. These data areprovided below.

The flagellar serotype of strain EG10327 was determined in thelaboratory of Dr. M. -M. Lecadet at the Pasteur Institute, Paris,France. The serotype of EG10327 was determined according to methodsdescribed by H. de Barjac (1981), and was found to be Bacillusthuringiensis kurstaki (H3a, 3b, 3c). Previously described B.thuringiensis strains containing cryIII-related genes were found to beserotype morrisoni (strain EG2158 containing cryIIIA); serotypetolworthi (strain EG2838 containing cryIIIB); and serotype kumamoloensis(strain EG4961 containing cryIIIB2) (Rupar et al., 1991). EG10327represents the first B. thuringiensis kurstaki strain that has beenshown to be toxic to coleopterans.

5.3 Example 3

Evaluation of the Crystal Proteins of EG10327

Strain EG10327 was further evaluated by characterizing the crystalproteins it produces. These studies were performed by growing EG10327 inDSG sporulation medium [0.8% (wt./vol.) Difco nutrient broth, 0.5%(wt./vol.) glucose, 10 mM K₂HPO₄, 10 mM KH₂PO₄, 1 mM Ca(NO₃)₂, 0.5 mMMgSO₄, 10 μM MnCl₂, 10 μM FeSO₄]. The sporulated culture containing bothspores and crystal proteins was then harvested by centrifugation andsuspended in deionized water. Crystal proteins were solubilized from thesuspension of EG10327 spores and crystals by incubating the suspensionin solubilization buffer [0.14 M Tris pH 8.0, 2% (wt./vol.) sodiumdodecyl sulfate (SDS), 5% (vol./vol.) 2-mercaptoethanol, 10% (vol./vol.)glycerol and 0.1% (wt./vol.) bromophenol blue] at 100° C. for 5 min.

The solubilized crystal proteins were size fractionated byelectrophoresis through an acrylamide gel (SDS-PAGE analysis). Aftersize fractionation, the proteins were visualized by staining withCoomassie dye. SDS-PAGE analysis showed that a major crystal protein ofapproximately 29 kDa, hereinafter referred to as the CryET33 protein,and a major crystal protein of approximately 14 kDa, hereinafterreferred to as the CryET34 protein, were solubilized from the sporulatedEG10327 culture.

The 29-kDa CryET33 protein and the 14-kDa CryET34 protein of EG10327were further characterized by determination of their NH₂-terminal aminoacid sequences as follows. The sporulated EG10327 culture was incubatedwith solubilization buffer and solubilized crystal proteins were sizefractionated through an acrylamide gel by SDS-PAGE analysis. Theproteins were transferred from the gel to a nitrocellulose filter bystandard electroblotting techniques. The CryET33 protein and the CryET34protein that had been electroblotted to the filter were visualized bystaining the filter with Coomassie dye. Portions of the filtercontaining the CryET33 protein and the CryET34 protein were excised witha razor blade. In this manner the CryET33 protein and the CryET34protein were obtained in pure forms as proteins blotted onto separatepieces of nitrocellulose filter.

The purified CryET33 and CryET34 proteins contained on pieces ofnitrocellulose filter were subjected to a standard automated Edmandegradation procedure in order to determine the NH₂-terminal amino acidsequence of each protein.

The NH₂-terminal sequence of the CryET33 protein of EG10327 was found tobe:

1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20(SEQ ID NO:5)GlyIleIleAsnIleGlnAspGluIleAsnAsnTyrMetLysGluValTyrGlyAlaThr

The NH₂-terminal sequence of the CryET34 protein of EG10327 was found tobe:

1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20(SEQ ID NO:6)ThrValTyrAsnValThrPheThrIleLysPheTyrAsnGluGlyGluTrpGlyGlyPro           (Ala)          (Asn)

The amino acid residues listed in parenthesis below the sequence of theCryET34 protein represent potential alternative amino acids that may bepresent in the CryET34 protein at the position indicated. Alternativeamino acids are possible due to the inherent uncertainty that exists inthe use of the automated Edman degradation procedure for determiningprotein amino acid sequences.

Computer algorithms (Korn and Queen, 1984) were used to compare theN-terminal sequences of the CryET33 and CryET34 proteins with amino acidsequences of all B. thuringiensis crystal proteins of which theinventors are aware including the sequences of all B. thuringiensiscrystal proteins which have been published in scientific literature,international patent applications, or issued patents. A list of thecrystal proteins whose sequences have been published along with thesource of publication is shown in Table 5.

TABLE 5 B. THURINGIENSIS CRYSTAL PROTEINS DESCRIBED IN THE LITERATURECrystal Protein Source or Reference Cry1A(a) J. Biol. Chem.,260:6264-6272 Cry1A(b) DNA, 5:305-314 Cry1A(c) Gene, 36:289-300 Cry1BNucl. Acids Res., 16:4168-4169 Cry1C Nucl. Acids Res., 16:6240 Cry1CbAppl. Environ. Micro., 59:1131-1137 Cry1C(b) Nucl. Acids Res., 18:7443Cry1D Nucl. Acids Res., 18:5545 Cry1E EPO 358 557 A2 Cry1F J.Bacteriol., 173:3966-3976 Cry1G FEBS, 293:25-28 CryV WO 90/13651 Cry2AJ. Biol. Chem., 263:561-567 Cry2B J. Bacteriol., 171:965-974 Cry2C FEMSMicrobiol. Lett., 81:31-36 Cry3A Proc. Natl. Acad. Sci. USA,84:7036-7040 Cry3B Nucl. Acids Res., 18:1305 Cry3B2 Appl. Environ.Microbiol., 58:3921-3927 Cry3B3 U.S. Pat. No. 5,378,625 Cry3C Appl.Environ. Microbiol., 58:2536-2542 Cry3D Gene, 110:131-132 Cry4A Nucl.Acids Res., 15:7195 Cry4B EPO 308,199 Cry4C J. Bacteriol., 166:801-811Cry4D J. Bacteriol., 170:4732, 1988 Cry5 Molec. Micro., 6:1211-1217Cry33AkD WO 94/13785 Cry33BkD WO 94/13785 Cry34kD J. Bacteriol.,174:549-557 Cry40kD J. Bacteriol., 174:549-557 Cry201T635 WO 95/02693Cry517 J. Gen. Micro., 138:55-62 Crya7A021 EPO 256,553 B1 CryAB78ORF1 WO94/21795 CryAB780RF2 WO 94/21795 CryAB78100kD WO 94/21795 Crybtpgs1208EPO 382 990 Crybtpgs 1245 EPO 382 990 Crybts02618A WO 94/05771 CryBuibuiWO 93/03154 CryET4 U.S. Pat. No. 5,322,687 CryET5 U.S. Pat. No.5,322,687 CryGei87 EPO 238,441 CryHD511 U.S. Pat. No. 5,286,486 CryHD867U.S. Pat. No. 6,286,486 CryIPL U.S. Pat. No. 5,231,008 CryMITS JP6000084 CryPS17A WO 92/19739 CryPS17B U.S. Pat. No. 5,350,576 and U.S.Pat. No. 5,424,410 CryP16 WO 95/00639 CryP18 WO 95/00639 CryP66 WO95/00639 CryPS33F2 WO 92/19739 and U.S. Pat. No. 5,424,410 CryPS40D1U.S. Pat. No. 5,273,746 CryPS43F WO 93/04587 CryPS 50Ca WO 93/04587 andEPO 498,537 A2 CryPS 50Cb WO 93/15206 Cryps52A1 U.S. Pat. No. 4,849,217CryPS63B WO 92/19739 CryPS69D1 U.S. Pat. No. 5,424,410 Cryps71M3 WO95/02694 CryPS80JJ1 WO 94/16079 CryPS81IA U.S. Pat. No. 5,273,746CryPS81IA2 EPO 405 810 Cryps81A2 EPO 401 979 CryPS81IB WO 93/14641CryPS81IB2 U.S. Pat. No. 5,273,746 Cryps81f U.S. Pat. No. 5,045,469Cryps81gg U.S. Pat. No. 5,273,746 Cryps81rr1 EPO 401 979 Cryps86A1 U.S.Pat. No. 5,468,636 CryX FEBS Lett., 336:79-82 CryXenA24 WO 95/00647CrycytA Nucl. Acids Res., 13:8207-8217

The N-terminal sequence of the CryET34 of EG10327 protein was not foundto be homologous to any of the known B. thuringiensis crystal proteinsidentified in Table 5.

5.4 Example 4

Characterization of the CryET33 Crystal Protein of EG2159

It had been previously determined that the 68-kDa CryIIIA protein ofEG2159 (referred to as the CryC protein in Donovan et al., 1988) wastoxic to Colorado potato beetle, but no protein had been identified inthe strain which had lepidopteran or dipteran activity.

Strain EG2159 was derived from B. thuringiensis strain EG2158 by curingof a 150-MDa plasmid from EG2158 (described in Donovan et al., 1988).EG2159 is identical to EG2158 except that EG2159 is missing a 150-MDaplasmid present in EG2158. One of the two crystal proteins produced byEG2159, the 68-kDa CryIIIA protein, was isolated, and the gene encodingit was cloned and sequenced. These results were described previously bythe inventors (Donovan et al., 1988). A minor protein species, a 29-kDaprotein of EG2159, was not further characterized.

This example describes the characterization of this 29-kDa CryET33crystal protein from B. thuringiensis EG2159.

5.4.1 Isolation of Crystal Proteins from EG2159

The crystal proteins of EG2159 were solubilized by suspending asporulated culture of EG2159 containing spores plus crystal proteins inprotein solubilization buffer at 80° C. for 3 min. The solubilizedcrystal proteins were size fractionated by SDS-PAGE and proteins in theSDS-PAGE gel were visualized by staining with Coomassie dye. Gel slicescontaining the 29-kDa protein were cut out of the SDS-PAGE gel with arazor blade and the protein was separated from the gel slices bystandard electroelution procedures. These studies resulted in a purifiedpreparation of the CryET33 protein from B. thuringiensis strain EG2159.

5.4.2 NH₂-Terminal Sequencing of the 29-kDa Protein

The NH₂-terminal amino acid sequence of the purified CryET33 protein wasdetermined by automated Edman degradation. The amino acid sequence ofthe NH₂-terminal portion of the 29-kDa protein was determined to be:

1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20MetGlyIleIleAsnIleGlnAspGluIleAsn--- (SEQ ID NO:7)TyrMetLysGluValTyrGlyAla (SEQ ID NO:8)

Dashes (---) at position 12 indicate that this amino acid residue couldnot be determined for the CryET33 protein of EG2159.

5.4.3 Results

Comparison of the sequence of the CryET33 protein of EG10327 (SEQ IDNO:5) and the NH₂-terminal sequence of the previously-uncharacterized29-kDa protein (Donovan et al., 1988) observed in EG2159 (SEQ ID NO:7,SEQ ID NO:8) suggested that the NH₂-terminal end of the 29-kDa proteinof EG2159 was identical to the CryET33 protein of EG10327, with theexception of an initial methionine (Met) residue present in the CryET33protein of EG2159.

5.5 Example 5

Isolation of a DNA Fragment Comprising cryET33 and cryET34 Genes fromEG2158

As described above, strain EG2159 was derived from strain EG2158.Therefore, EG2158 contains the identical gene for the CryET33 protein,hereinafter referred to as the cryET33 gene, as strain EG2159. To clonethe cryET33 gene reverse genetics was used. A 33-mer oligonucleotidcprobe (designated WD68) encoding amino acids 1 through 11 of theNH₂-terminus of the CryET33 protein was synthesized. The sequence ofWD68 is:

5′-ATGGGAATTATTAATATTCAAGATGAAATTAAT-3′ (SEQ ID NO:9)

WD68 was used as a probe in Southern hybridization studies as describedbelow in attempts to identify a DNA fragment from EG2158 that containedthe cryET33 gene for the 29-kDa CryET33 protein. Total DNA was extractedfrom EG2158 by a standard lysozyme/phenol method. The extracted DNA wasdigested with DNA restriction enzymes HindIII and EcoRI, and thedigested DNA was size fractionated by electrophoresis through an agarosegel. The DNA fragments were blotted from the gel to a nitrocellulosefilter using previously described methods (Southern, 1975), and thefilter was incubated with oligonucleotide WD68 that had beenradioactively labeled with T4 kinase and [γ-³²P]ATP. No unique DNArestriction fragment from EG2158 was found to which the WD68 probespecifically hybridized.

A different approach was then used to identify a DNA restrictionfragment that contained the cryET33 gene. A 56-mer oligonucleotide probe(designated WD73) encoding amino acids 1 through 19 of the NH₂-terminusof the CryET33 protein was synthesized. The sequence of WD73 is:

5′-ATGGGAATTATTAATATTCAAGATGAAATTAATNNNTATATGAAAGAAGTATATGG-3′ (SEQ IDNO:10)

where the three N's corresponding to amino acid 12 of WD73 representthree inosine nucleotides. Inosine residues were used at this positionto encode the corresponding unknown amino acid at position 12 in theNH₂-terminal sequence of the CryET33 protein. Inosine is considered tobe a neutral nucleotide, and neither promotes nor hinders binding of DNAstrands. WD73 was radioactively labeled with T4 kinase and [γ-³²P]ATPand used to probe a nitrocellulose filter containing size-fractionatedHindIII and EcoRI restriction fragments of EG2158 total DNA. WD73specifically hybridized to a HindIII fragment of approximately 7.9 kb,and to an EcoRI fragment of approximately 5.2-kb of EG2158 DNA.

5.6 Example 6

Cloning of the cryET33 and cryET34 Genes from EG2158

To isolate the 5.2-kb EcoRI fragment described in the previous Example,a plasmid library of strain EG2158 was constructed by ligatingsize-selected DNA EcoRI restriction fragments from strain EG2158 intothe E. coli vector pBR322. This procedure involved first obtaining totalDNA from strain EG2158 by cell lysis followed by phenol extraction ofDNA, then digesting the total DNA with EcoRI restriction enzyme,electrophoresing the digested DNA through an agarose gel, excising a gelslice containing EcoRI DNA fragments ranging in size from approximately4.0 to 6.0 kb, and electroeluting the size selected EcoRI restrictionfragments from the agarose gel slice. These fragments were mixed withthe E. coli plasmid vector pBR322, which had also been digested withEcoRI. The pBR322 vector carries the gene for Amp^(R) and the vectorreplicates in E. coli. T4 DNA ligase and ATP were added to the mixtureof size-selected restriction fragments of DNA from strain EG2158 and ofdigested pBR322 vector to allow the pBR322 vector to ligate with strainEG2158 restriction fragments.

The plasmid library was then transformed into E. coli cells, a hostorganism lacking the cryET33 and cryET34 genes of interest as follows.After ligation, the DNA mixture was incubated with an Amp^(S) E. colihost strain, HB101, that had been made competent using standard CaCl₂procedures. E. coli HB101, was used as the host strain because thesecells are easily transformed with recombinant plasmids and because HB101does not naturally contain genes for B. thuringiensis crystal proteins.Since pBR322 expresses Amp^(R), all host cells acquiring a recombinantplasmid were Amp^(R). After transforming host cells with the recombinantplasmids, cells were spread on agar medium that contained Amp. Afterincubation overnight at a temperature of 37° C., several thousand E.coli colonies grew on the Amp-containing agar, and these colonies werethen blotted onto nitrocellulose filters for subsequent probing.

The radioactively-labeled oligonucleotide WD73 was then used as a DNAprobe under conditions that permitted the probe to bind specificallythose transformed host colonies that contained the 5.2-kb EcoRI fragmentof DNA from strain EG2158. Several E. coli colonies specificallyhybridized to the WD73 probe. One WD73-hybridizing colony, designated E.coli EG11460, was studied further. E. coli EG11460 contained arecombinant plasmid, designated pEG246, which consisted of pBR322 plusthe inserted EcoRI restriction fragment of DNA from strain EG2158 ofapproximately 5.2 kb. A restriction map of pEG246 is shown in FIG. 2.The E. coli strain EG11460 containing pEG246 has been deposited with theAgricultural Research Culture Collection, Northern Regional ResearchLaboratory (NRRL) under the terms of the Budapest Treaty havingAccession No. NRRL B-21364.

The nucleotide base sequence of approximately one-third of the cloned5.2-kb EcoRI fragment of pEG246 was determined using the standard Sangerdideoxy method. Sequencing revealed that the 5.2-kb fragment containedtwo adjacent open reading frames encoding proteins and, in particular,two novel crystal toxin genes. The upstream open reading frame,designated cryET33, encoded a protein whose NH₂-terminal sequencematched the NH₂-terminal sequence of the 29-kDa CryET33 protein ofstrains EG2159 and EG10327. The downstream gene, designated cryET34,encoded a protein whose amino acid sequence matched the NH₂-terminalamino acid sequence determined for the 14 kDa CryET34 protein ofEG10327. The DNA sequences of these new genes are significantlydifferent from the sequences of the known crystal toxin genes of B.thuringiensis listed in Table 5.

The DNA sequence of the cryET33 gene (SEQ ID NO:1) and the deduced aminoacid sequence of the CryET33 protein (SEQ ID NO:3) encoded by thecryET33 gene are shown in FIG. 1A, FIG. 1B, and FIG. 1C. The proteincoding portion of the cyET33 gene (SEQ ID NO:1) is defined by thenucleotides starting at position 136 and ending at position 936. Thesize of the CryET33 protein (SEQ ID NO:3) as deduced from the cryET33gene (SEQ ID NO:1) is 29,216 Da (267 amino acids). Also shown in FIG.1A, FIG. 1B, and FIG. 1C are the DNA sequence of the cryET34 gene (SEQID NO:2) and the deduced amino acid sequence of the CryET34 protein (SEQID NO:4) encoded by the cryET34 gene. The protein coding portion of thecryET34 gene (SEQ ID NO:2) is defined by the nucleotides starting atposition 969 and ending at position 1346. The size of the CryET34protein (SEQ ID NO:4) as deduced from the cryET34 gene (SEQ ID NO:2) is14,182 Da (126 amino acids).

Computer algorithms (Korn and Queen, 1984; Altschul et al., 1990) wereused to compare the DNA sequences of the cryET33 and cryET34 genes andthe deduced amino acid sequences of the CryET33 and CryET34 proteins tothe sequences of all B. thuringiensis cry genes and crystal proteins ofwhich the inventors are aware (described in section 5.3, Example 3, andlisted in Table 5) and to the sequences of all genes and proteinscontained in the Genome Sequence Data Base (National Center for GenomeResources, Santa Fe, N.Mex.). The sequence of the cryET34 gene (SEQ IDNO:2) and the deduced sequence of the CryET34 protein (SEQ ID NO:4) werenot found to be related to any known genes or proteins, respectively.The sequence of the cryET33 gene (SEQ ID NO:1) was found to havesequence identity with only one known gene and the sequence identity wasvery low. The sequence of the cryET33 gene (801 nucleotides) was 38%identical with the sequence of a B. thuringiensis subsp. thompsoni gene(1,020 nucleotides) described by Brown and Whiteley (1992). The deducedsequence of the CryET33 protein (SEQ ID NO:3) was found to have sequenceidentity with only one known protein and the identity was very low. Thecomplete amino acid sequence of the CryET33 protein (267 amino acids)was found to be 27% identical with the complete amino acid sequence of aB. thuringiensis subsp. thompsoni crystal protein (340 amino acids)described by Brown and Whiteley, 1992 for a caterpillar-toxic protein.

The DNA sequence immediately upstream from the cryET33 gene (FIG. 1A,FIG. 1B, and FIG. 1C, nucleotides 1 to 135) was searched for homologieswith all known upstream DNA sequences of crystal protein genes and withthe DNA sequences of all known genes in the Genomc Sequence Database(Table 5). DNA sequences immediately upstream from coding regions ofgenes often contain promoters for expression of the corresponding genes.This search resulted in no homologies being found.

5.7 Example 7

Expression of Recombinant cryET33 and cryET34 Genes

Experience has shown that cloned B. thuringiensis crystal toxin genesare poorly expressed in E. coli but are often highly expressed inrecombinant B. thuringiensis strains. pEG246, containing the cryET33 andcryET34 genes (FIG. 2), is capable of replicating in E. coli but not inB. thuringiensis. To obtain a plasmid containing the cryET33 and cryET34genes and capable of replicating in B. thuringiensis, a Bacillus spp.plasmid was inserted into pEG246 as described below.

The Bacillus spp. plasmid pNN101 (Norton et al., 1985) capable ofreplicating in B. thuringiensis and conferring chloramphenicolresistance (Cam^(R)) and tetracycline resistance (Tet^(R)) was digestedwith BamHI and the digested plasmid was mixed with plasmid pEG246 thathad been digested with BamHI. The two plasmids were ligated togetherwith T4 ligase plus ATP. The ligation mixture was then used to transformcompetent E. coli DH5α cells. After incubation with the plasmid mixturethe cells were plated on agar plates containing Tet. It was expectedthat cells which had taken up a plasmid consisting of pNN101 ligatedwith pEG246 would be Tet^(R). After incubation for approximately 20 hrseveral Tet^(R) E. coli colonies grew on the agar plates containing Tet.

Plasmid DNA was isolated from one Tet^(R) colony. The plasmid wasdigested with BamHI, and electrophoresed through an agarose gel. Theplasmid, which was designated pEG1246, consisted of two BamHI DNAfragments of 5.8 kb and 9.6 kb corresponding to plasmids pNN101 andpEG246, respectively. A restriction map of pEG1246 is shown in FIG. 3.

B. thuringiensis strain EG10368 was then transformed by electroporationwith pEG1246 using previously described methods (Macaluso and Mettus,1991). Untransformed host cells of EG10368 are crystal negative (Cry)and Cam^(S). After electroporation, the transformation mixture wasspread onto an agar medium containing Cam and were incubatedapproximately 16 hr at 30° C. pEG1246-transformed cells would Cam^(R).One Cam^(R) colony, designated B. thuringiensis strain EG11403,contained a plasmid whose restriction pattern was identical to that ofpEG1246.

Cells of strain EG11403 were grown in DSG sporulation medium containingCam at 22° C. to 25° C. until sporulation and cell lysis had occurred(4-5 days). Microscopic examination revealed that the sporulated cultureof strain EG11403 contained spores and small free floatingspindle-shaped and irregularly shaped crystals. The crystals resembledthose observed with a sporulated culture of strain EG10327.

Spores, crystals and cell debris from the sporulated culture of strainEG11403 were harvested by centrifugation. The centrifuge pellet waswashed once with deionized water, and the pellet suspended in deionizedwater.

Crystal proteins in the EG11403 suspension were characterized bysolubilization and SDS-PAGE analysis. SDS-PAGE analysis revealed thatstrain EG11403 produced two major proteins of 29 kDa and 14 kDa. Asexpected the 29 kDa protein and the 14-kDa protein of strain EG11403were identical in size to the 29-kDa CryET33 protein and to the 14-kDaCryET34 protein, respectively, produced by strain EG10327. StrainEG11403 was deposited on Dec. 14, 1994, with the Agricultural ResearchCulture Collection, Northern Regional Research Laboratory (NRRL) underthe terms of the Budapest Treaty having Accession No. NRRL B-21367.

The gene encoding the 29 kDa CryET33 protein of EG11403 is the cryET33gene and the gene encoding the 14 kDa CryET34 protein of EG11403 is thecryET34 gene. B. thuringiensis strains EG11403 and EG10327 producedapproximately equal amounts of CryET33 protein. In contrast, B.thuringiensis strain EG2158 produced approximately {fraction (1/10)}ththe amount of the CryET33 protein as either strain EG11403 or strainEG10327.

5.8 Example 8

B. thuringiensis EG11402 Containing CryIIIB3, CryET33 and CryET34

It was previously shown that the B. thuringiensis crystal proteindesignated as CryIIIB3 was toxic to larvae of the Japanese beetle (U.S.Pat. No. 5,264,364). In the following example, the CryET33 and CryET34proteins were found to be toxic to boll weevil, and Japanese beetlelarvae. The Cry3B3 protein shares no amino acid sequence homology witheither the CryET33 protein or the CryET34 protein. In an attempt toproduce a strain having enhanced Japanese beetle toxicity the cry3B3gene. the cryET33 gene, and the cryET34 gene were combined in one strainas follows.

Strain EG10364 is a wild-type B. thuringiensis strain containing thecryIIIB3 gene. EG10364 produces the Japanese beetle larvae-toxic Cry3B3protein. pEG1246 (FIG. 3) containing the cryET33 and cryET34 genes wasused to transform EG10364 by electroporation to give rise to strainEG11402. EG11402 is identical to EG10364 except that EG11402 alsocontains pEG1246 (bearing the cloned cryET33 and cryET34 genes), and isconsequently Cam^(R).

Strain EG11402 was grown in DSG sporulation medium plus Cam at roomtemp. until sporulation and cell lysis occurred (4-5 days). Crystalproteins were solubilized from the sporulated EG11402 culture and thesolubilized proteins were size fractionated by SDS-PAGE. This analysisrevealed that strain EG11402 produced three crystal proteins: a 70-kDacrystal protein corresponding to the CryIIIB3 protein, a 29-kDa crystalprotein corresponding to the CryET33 protein, and a 14-kDa crystalprotein corresponding to the CryET34 protein. SDS-PAGE analysis showedthat strain EG10364, which had been grown in an identical manner asEG11402 except without chloramphenicol, produced the 70-kDa CryIIIB3protein in similar amounts as EG11402. Strain EG11402 was deposited onDec. 14, 1994 under the terms of the Budapest Treaty with theAgricultural Research Culture Collection, Northern Regional ResearchLaboratory (NRRL) having Accession No. NRRL B-21366.

5.9 Example 9

Toxicity of CryET33 and CryET34 to Japanese Beetle Larvae

The toxicity to Japanese beetle larvae (Popillia japonica) wasdetermined for three B. thuringiensis strains: (1) strain EG10327producing the CryET33 and CryET34 crystal proteins; (2) strain EG10364producing the Cry3B3 crystal protein; and (3) strain EG11402 producingthe CryET33, CryET34 and Cry3B3 crystal proteins.

Strains EG10327, EG10364, and EG11402 were grown in DSG sporulationmedium at room temperature (20 to 23° C.) until sporulation and celllysis had occurred (4-5 days). For EG11402, the medium contained 5 μg/mlCam. The fermentation broth was concentrated by centrifugation and thepellets, containing spores, crystal proteins and cell debris were eitherfreeze dried to yield powders or were resuspended in deionized water toyield aqueous suspensions. The amounts of the Cry3B3 and CryET33 crystalproteins in the freeze-dried powders and in the suspensions werequantified using SDS-PAGE techniques and densitometer tracing ofCoomassie stained SDS-PAGE gels with purified and quantified Cry3Aprotein as a standard. The amount of the CryET34 protein was estimatedby visual inspection of Coomassie stained SDS-PAGE gels. This inspectionindicated that the amount of the CryET34 protein was roughly equivalentto the amount of the CryET34 protein in strains EG10327 and EG11402.

The bioassay procedure for Japanese beetle larvae was carried out asfollows. freeze-dried powders of each strain to be tested were suspendedin a diluent (an aqueous solution containing 0.005% Triton X-100®) andwere incorporated into 100 ml of hot (50-60° C.) liquid artificial diet,based on the insect diet previously described (Ladd, 1986). The mixtureswere allowed to solidify in Petri dishes, and 19-mm diameter plugs ofthe solidified diet were then placed into ⅝ ounce plastic cups. OneJapanese beetle larvae was introduced into each cup, the cups werecovered with a lid and held at 25° C. for fourteen days before larvaemortality was scored. Two replications of sixteen larvae each werecarried out in this study.

The results of this toxicity test are shown below in Table 6, whereinsecticidal activity is reported as percentage of dead larvae, with thepercent mortality being corrected for control death, the control beingdiluent only incorporated into the diet plug.

TABLE 6 ACTIVITY OF CRYET33, CRYET34 AND CRY3B3 TO JAPANESE BEETLELARVAE Strain Protein(s) Present Protein Dose Insect Mortality EG10327CryET33 ˜4,000 ppm 95% CryET34  ND^(a) EG10364 Cry3B3 500 ppm 38%EG11402 Cry3B3 560 ppm 58% CryET33 ˜1,000 ppm CryET34 ND ^(a)ND, notdetermined.

The results shown in Table 6 demonstrate that the CryET33 and CryET34proteins have significant toxicity to Japanese beetle larvae. EG10327,which produces the CryET33 and CryET34 proteins, is toxic to Japanesebeetle larvae. EG10364 which produces the Cry3B3 protein is also toxicto Japanese beetle larvae. When the cryET33 and cryET34 genes are addedto EG10364, resulting in EG11402 which produces the CryET33 and CryET34proteins in addition to the Cry3B3 protein, an enhanced toxicity toJapanese beetle larvae was seen.

5.10 Example 10

Toxicity of CryET33 and CryET34 to Red Flour Beetle Larvae

The toxicity to red flour beetle larvae (Tribolium castaneum) wasdetermined for four B. thuringiensis strains: (1) EG10327 producing theCryET33 and CryET34 crystal proteins; (2) EG10364 producing the Cry3B3crystal protein; (3) EG11403 producing the CryET33 and CryET34 crystalproteins: and (4) EG11402 producing the Cry3B3, CryET33 and CryET34crystal proteins. The four strains were grown in DSG medium untilsporulation and cell lysis had occurred, and aqueous suspensions orfreeze dried powders were prepared as described in Example 9. Thetoxicity of each strain against red flour beetle larvae was determinedby applying a known amount of each strain preparation to an artificialdiet and feeding the diet to red flour beetle larvae.

The results of this toxicity test are shown in Table 7, whereinsecticidal activity is reported as percentage insect mortality, withthe mortality being corrected for control death, the control beingdiluent only incorporated into the diet.

TABLE 7 TOXICITY OF CRYET33, CRYET34 AND CRY3B3 PROTEINS TO RED FLOURBEETLE LARVAE Strain Protein Protein Dose Insect Mortality EG10327CryET33 ˜2,000 ppm 100%  CryET34  ND^(a) EG10364 Cry3B3 448 ppm 74%EG11402 Cry3B3 448 ppm 97% CryET33 ˜2,000 ppm CryET34 ND EG11403 CryET33˜2,000 ppm 39% CryET34 ND ^(a)ND, not determined

The results shown in Table 7 demonstrate that the CryET33 and CryET34proteins have a significant level of toxicity to red flour beetlelarvae. The naturally occurring strain EG10327 which produces theCryET33 and CryET34 proteins is highly toxic to red flour beetle larvae.EG10364 which produces the Cry3B3 protein is toxic to red flour beetlelarvae. EG11403 which produces the CryET33 and the CryET34 proteins istoxic to red flour beetle larvae. When the cryET33 and cryET34 genes areadded to EG10364, giving rise to EG11402, an enhanced toxicity to redflour beetle larvae is seen in the resultant strain which producesCryET33, CryET34, and Cry3B3 proteins.

5.11 Example 11

Toxicity of CryET33 and CryET34 on Boll Weevil Larvae

EG 11403 producing the CryET33 and Cry ET34 proteins was grown asdescribed. The protein crystal was washed, solubilized in carbonatebuffer, dialyzed and filtered through a 0.2 U acrodisc. The toxicity ofthe solubilized proteins were then determined by adding a known amountof the proteins to artificial diet and feeding the diet to boll weevillarvae. The results of this toxicity test are shown below, whereinsecticidal activity is reported as either (1) percent mortality, withthe mortality being corrected for control death using a buffer control;or (2) percent mortality+the percent of larvae not developing beyondfirst instar, with the mortality again being corrected for control deathusing a buffer control.

The results in Table 8 and Table 9 demonstrate that Cry ET33 and CryET34proteins have a significant level of toxicity to boll weevil larvae.

TABLE 8 (1) BOLL WEEVIL PERCENT MORTALITY μg/ml % Mortality 40 50 2046.67 10 11.76 5 6.67 2.5 0 1.25 6.67 0.31 0 0.08 10

TABLE 9 (2) PERCENT MORTALITY + 1ST INSTARS μg/ml % mortality + 1^(st)instar 40 100 20 93.33 10 64.71 5 40 2.5 11.11 1.25 6.67 0.31 5.88 0.0810

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All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecomposition, methods and in the steps or in the sequence of steps of themethod described herein without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims. Accordingly, the exclusive rights sought to be patentedare as described in the claims below.

10 807 base pairs nucleic acid single linear not provided 1 ATGGGAATTATTAATATCCA AGATGAAATT AATAATTACA TGAAAGAGGT ATATGGTGCA 60 ACAACTGTTAAAAGCACATA CGATCCCTCA TTCAAAGTAT TTAATGAATC TGTGACACCC 120 CAATTCACTGAAATTCCAAC AGAACCTGTA AATAATCAAT TAACTACAAA AAGAGTAGAT 180 AATACGGGTAGTTACCCAGT AGAAAGTACT GTATCGTTCA CATGGACGGA AACCCATACA 240 GAAACAAGTGCAGTAACTGA GGGAGTGAAA GCCGGCACCT CAATAAGTAC TAAACAATCT 300 TTTAAATTTGGTTTTGTTAA CTCTGATGTT ACTTTAACGG TATCAGCAGA ATATAATTAT 360 AGTACAACAAATACAACTAC AACAACAGAA ACACACACCT GGTCAGATTC AACAAAAGTA 420 ACTATTCCTCCCAAAACTTA TGTGGAGGCT GCATACATTA TCCAAAATGG AACATATAAT 480 GTTCCGGTTAATGTAGAATG TGATATGAGT GGAACTTTAT TTTGTAGAGG GTATAGAGAT 540 GGTGCGCTTATTGCAGCAGT TTATGTTTCT GTAGCGGATT TAGCAGATTA CAATCCAAAT 600 TTAAATCTTACAAATAAAGG GGATGGAATT GCTCACTTTA AAGGTTCGGG TTTTATAGAG 660 GGTGCACAAGGCTTGCGAAG CATTATTCAG GTTACAGAAT ATCCACTAGA TGATAATAAA 720 GGTCGCTCGACACCAATAAC TTATTTAATA AATGGTTCAT TAGCACCAAA TGTTACATTA 780 AAAAATAGCAACATAAAATT TTAATAA 807 381 base pairs nucleic acid single linear notprovided 2 ATGACAGTAT ATAACGCAAC TTTCACCATT AATTTCTATA ATGAAGGAGAATGGGGGGGG 60 CCAGAACCAT ATGGTTATAT AAAAGCATAT CTTACAAATC CAGATCATGATTTTGAAATT 120 TGGAAACAAG ATGATTGGGG GAAAAGTACT CCTGAGAGAA GTACTTATACGCAAACGATT 180 AAAATAAGTA GCGACACTGG TTCCCCTATA AACCAAATGT GTTTTTATGGTGATGTGAAA 240 GAATACGACG TAGGAAATGC AGATGATATT CTCGCTTATC CAAGTCAAAAAGTATGCAGT 300 ACACCTGGTG TAACAGTACG ACTTGATGGC GATGAGAAAG GTTCTTATGTGACAATTAAG 360 TATTCCTTGA CTCCAGCATA A 381 267 amino acids amino acidsingle linear not provided 3 Met Gly Ile Ile Asn Ile Gln Asp Glu Ile AsnAsn Tyr Met Lys Glu 1 5 10 15 Val Tyr Gly Ala Thr Thr Val Lys Ser ThrTyr Asp Pro Ser Phe Lys 20 25 30 Val Phe Asn Glu Ser Val Thr Pro Gln PheThr Glu Ile Pro Thr Glu 35 40 45 Pro Val Asn Asn Gln Leu Thr Thr Lys ArgVal Asp Asn Thr Gly Ser 50 55 60 Tyr Pro Val Glu Ser Thr Val Ser Phe ThrTrp Thr Glu Thr His Thr 65 70 75 80 Glu Thr Ser Ala Val Thr Glu Gly ValLys Ala Gly Thr Ser Ile Ser 85 90 95 Thr Lys Gln Ser Phe Lys Phe Gly PheVal Asn Ser Asp Val Thr Leu 100 105 110 Thr Val Ser Ala Glu Tyr Asn TyrSer Thr Thr Asn Thr Thr Thr Thr 115 120 125 Thr Glu Thr His Thr Trp SerAsp Ser Thr Lys Val Thr Ile Pro Pro 130 135 140 Lys Thr Tyr Val Glu AlaAla Tyr Ile Ile Gln Asn Gly Thr Tyr Asn 145 150 155 160 Val Pro Val AsnVal Glu Cys Asp Met Ser Gly Thr Leu Phe Cys Arg 165 170 175 Gly Tyr ArgAsp Gly Ala Leu Ile Ala Ala Val Tyr Val Ser Val Ala 180 185 190 Asp LeuAla Asp Tyr Asn Pro Asn Leu Asn Leu Thr Asn Lys Gly Asp 195 200 205 GlyIle Ala His Phe Lys Gly Ser Gly Phe Ile Glu Gly Ala Gln Gly 210 215 220Leu Arg Ser Ile Ile Gln Val Thr Glu Tyr Pro Leu Asp Asp Asn Lys 225 230235 240 Gly Arg Ser Thr Pro Ile Thr Tyr Leu Ile Asn Gly Ser Leu Ala Pro245 250 255 Asn Val Thr Leu Lys Asn Ser Asn Ile Lys Phe 260 265 126amino acids amino acid single linear not provided 4 Met Thr Val Tyr AsnAla Thr Phe Thr Ile Asn Phe Tyr Asn Glu Gly 1 5 10 15 Glu Trp Gly GlyPro Glu Pro Tyr Gly Tyr Ile Lys Ala Tyr Leu Thr 20 25 30 Asn Pro Asp HisAsp Phe Glu Ile Trp Lys Gln Asp Asp Trp Gly Lys 35 40 45 Ser Thr Pro GluArg Ser Thr Tyr Thr Gln Thr Ile Lys Ile Ser Ser 50 55 60 Asp Thr Gly SerPro Ile Asn Gln Met Cys Phe Tyr Gly Asp Val Lys 65 70 75 80 Glu Tyr AspVal Gly Asn Ala Asp Asp Ile Leu Ala Tyr Pro Ser Gln 85 90 95 Lys Val CysSer Thr Pro Gly Val Thr Val Arg Leu Asp Gly Asp Glu 100 105 110 Lys GlySer Tyr Val Thr Ile Lys Tyr Ser Leu Thr Pro Ala 115 120 125 20 aminoacids amino acid single linear not provided 5 Gly Ile Ile Asn Ile GlnAsp Glu Ile Asn Asn Tyr Met Lys Glu Val 1 5 10 15 Tyr Gly Ala Thr 20 20amino acids amino acid single linear not provided 6 Thr Val Tyr Asn ValThr Phe Thr Ile Lys Phe Tyr Asn Glu Gly Glu 1 5 10 15 Trp Gly Gly Pro 2011 amino acids amino acid single linear not provided 7 Met Gly Ile IleAsn Ile Gln Asp Glu Ile Asn 1 5 10 8 amino acids amino acid singlelinear not provided 8 Tyr Met Lys Glu Val Tyr Gly Ala 1 5 33 base pairsnucleic acid single linear not provided 9 ATGGGAATTA TTAATATTCAAGATGAAATT AAT 33 56 base pairs nucleic acid single linear not providedmodified_base 34..36 /mod_base= OTHER /note= “N = Inosine” 10 ATGGGAATTATTAATATTCA AGATGAAATT AATNNNTATA TGAAAGAAGT ATATGG 56

What is claimed is:
 1. A composition comprising an insecticidallyeffective amount of a first polypeptide and a second polypeptide,wherein said first polypeptide comprises an amino acid sequence at least80% identical to SEQ ID NO:4, and said second polypeptide comprises anamino acid sequence at least 80% identical to SEQ ID NO:3; and anagriculturally acceptable carrier, wherein the composition issubstantially free of intact cells.
 2. The composition of claim 1,wherein the first polypeptide comprises an amino acid sequence at least95% identical to SEQ ID NO:4.
 3. The composition of claim 2, wherein thefirst polypeptide comprises SEQ ID NO:4.
 4. The composition of claim 1,comprising from about 1% to about 50% by weight of the firstpolypeptide.
 5. The composition of claim 1, wherein the firstpolypeptide comprises an amino acid sequence encoded by a nucleic acidat least 90% identical to SEQ ID NO:2.
 6. The composition of claim 1,wherein the first polypeptide comprises an amino acid sequence encodedby a nucleic acid at least 95% identical to SEQ ID NO:2.
 7. Thecomposition of claim 1, wherein the first polypeptide comprises an aminoacid sequence encoded by SEQ ID NO:2.
 8. The composition of claim 1,wherein the second polypeptide is at least 80% identical to SEQ ID NO:3.9. The composition of claim 1, wherein the second polypeptide is atleast 95% identical to SEQ ID NQ:3.
 10. The composition of claim 1,wherein the second polypeptide is SEQ ID NO:3.